KR101650995B1 - Cxcr2 binding polypeptides - Google Patents

Abstract

The present invention relates to polypeptides which act on or specifically bind to the chemokine receptor CXCR2 and, in particular, polypeptides capable of regulating signal transduction from CXCR2. The present invention also relates to nucleic acids, vectors and host cells capable of expressing the polypeptides of the invention, pharmaceutical compositions comprising the polypeptides and the use of said polypeptides and compositions for the treatment of diseases involving abnormal function of CXCR2.

Description

CXCR2 binding polypeptides {CXCR2 BINDING POLYPEPTIDES}

The present invention relates to polypeptides which act on or specifically bind to the chemokine receptor CXCR2 and, in particular, polypeptides capable of regulating signal transduction from CXCR2. The present invention also relates to nucleic acids, vectors and host cells capable of expressing the polypeptides of the present invention, pharmaceutical compositions comprising the polypeptides, and polypeptides of the invention for the treatment of chronic obstructive pulmonary disease (COPD) and other diseases involving abnormal function of CXCR2 And to the use of the composition.

BACKGROUND OF THE INVENTION [0002] Chronic obstructive pulmonary disease (COPD) is a term that is used in many cases to describe various disorders characterized by airflow limitation associated with abnormal inflammatory response of the lungs to harmful particles with destruction of pulmonary parenchyma, [Barnes PJ et al., 2004, Mediators of chronic obstructive pulmonary disease, Pharmacol (2004), Chronic obstructive pulmonary disease: molecular and cellular mechanisms, Eur. Respir J, 22, 672-688; Rev. 56, 515-548). Although genetic and environmental factors contribute to the onset of COPD, smoking is the single most important cause, and recurrent pulmonary infections lead to progressive reductions in pulmonary function. Smoking cessation reduces disease progression only when initially applied, and is less effective after significant symptoms occur. Several co-morbid conditions, such as asthma, cardiovascular disease, depression, and muscle weakness, are associated with COPD (Mannino DM and Buist S, 2007. Global burden of COPD: risk factors, prevalence and future trends. Lancet, 370, 765-773).

Chemokines are predominantly present in chemotactic factors and thus play a key role in the regulation of chronic inflammation of the COPD lung and its further enlargement during acute exacerbation. The biological activity of chemokine IL-8 (CXCL8), GROα (CXCL1) and ENA-78 (CXCL5) is mediated by two groups of cell-surface receptors CXCR1 and CXCR2, which are present on white blood cells and many other cell types throughout the body Mediated. Leukocyte migration is primarily mediated through CXCR2 binding to several ligands, including IL-8, GROa, beta, gamma, ENA78, and GCP-2. In contrast, CXCR1 is selectively activated by IL-8 and to a lesser extent by GCP-2. Whether human neutrophil chemotaxis in vivo is mediated by either a single receptor or both receptors remains unclear.

CXCR2 shares 78% homology at amino acid level with CXCR1, and both receptors are present on neutrophils in different distribution patterns. Expression of CXCR2 on a variety of cells and tissues, including multiple cell types within CD8 + T cells, NK, monocytes, mast cells, epithelium, endothelium, smooth muscle, and central nervous system, suggests that the receptors underlie constitutive conditions and many acute and chronic diseases Suggesting that it can perform a wide functional role in the pathophysiology of the disease. CXCR2 activation stimulates the binding of the Gi family of guanine nucleotide-binding proteins to the receptor, which in turn leads to the release of intracellular inositol phosphate, increase in intracellular Ca < ^{2 +} > and induction of a chemokine gradient by an ERK1 / 2-dependent mechanism Stimulates phosphorylation of intracellular proteins associated with cell migration. Once activated, CXCR2 is phosphorylated and rapidly internalized via arrestin / dynamin-dependent mechanisms, leading to receptor desensitization. This process is similar to that observed in most other GPCRs, but the ratio and extent of agonist-induced internalization of CXCR2 is greater than that observed in CXCR1 (Richardson RM, Pridgen BC, Haribabu B, Ali H, Synderman R. 1998 Differential cross-regulatory of the human chemokine receptors CXCR1 and CXCR2. Evidence for time-dependent signal generation. J Biol. Chem., 273, 23830-23836).

IL-8 has been proposed for a long time as a mediator of HCV-mediated inflammation in COPD (Keatings VM et al., 1996, Differences in IL-8 and tumor necrosis factor-α in induced sputum from patients with COPD and asthma. Respir. Crit. Care Med. 153, 530-534]; [Yamamoto C et al., 1997 Airway inflammation in COPD assessed by sputum levels of interleukin-8. There is an increase in T cell infiltration and neutrophil counts in the bronchial airways, small airways and pulmonary parenchyma from COPD patients, especially in the airway lumen (Hogg JC et al., 2004, The nature of small- airway obstruction in chronic obstructive pulmonary disease, N Eng. J. Med., 350, 2645-2653). Neutrophils increase in the lungs of patients with COPD, which is associated with disease severity (Keatings VM et al., 1996, Differences in IL-8 and tumor necrosis factor-α in induced sputum from patients with COPD and asthma. Respir Crit Care Med., 153, 530-534). In addition, the level of TNF [alpha] increases in the spleen of COPD patients, which induces IL-8 from airway epithelial cells (Keatings). GROα concentrations are significantly increased in COPD-induced sputum and bronchoalveolar lavage (BAL) fluids compared to normal smokers and non-smokers (Traves SL et al 2002, Increased levels of the chemokines GROα and MCP-1 in sputum et al., 1998, Inflammatory cells and mediators in bronchial lavage of patients with COPD, Eur Respir J. 12, 380-386). GROα is secreted by alveolar macrophages and airway epithelial cells in response to TNFα stimulation and selectively activates chemotactic CXCR2 for neutrophils and monocytes. There is an increase in monocyte chemotactic response to GROα in COPD patients, which may be associated with increased replacement or recirculation of CXCR2 in these cells (Traves SL et al., 2004, Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2, J. Leukoc. Biol. 76, 441-450). Viral and bacterial lung infections often lead to severe deterioration of COPD patients characterized by increased neutrophil counts in airways (Wedzicha JA, Seemungal TA, 2007, COPD exacerbations: defining their cause and prevention, Lancet 370 (9589) : 786-96). Bronchial biopsy of patients with severe acute exacerbation of COPD showed significantly increased amounts of ENA-78, IL-8 and CXCR2 mRNA expression (Qiu Y et al., 2003, Biopsy neutrophilia, neutrophil chemokine and receptor gene expression 168, 968-975), sputum showed an increased neutrophil count (Bathoorn E, Liesker JJw, and Postma DS et al. (2009) Int J COPD, 4 (1): 101-9), which suggests that the potential of the receptor in both COPD and severe aggravation of the disease Suggest a role. Increased expression of CXCR2 mRNA is seen in bronchial biopsy specimens, which is associated with the presence of tissue neutrophils (Qiu 2003). ENA-78 is predominantly derived from epithelial cells and there is a significant increase in ENA-78 expression in epithelial cells during the deterioration of COPD (Qiu 2003). Since the concentrations of IL-8, GROa and ENA-78 are increased in COPD airways and all three ligands transmit signals through CXCR2, blockade of the common receptor with selective antagonists is an effective antiinflammatory strategy in the disease Will be.

COPD progresses slowly and progressively, and disease progression is traditionally assessed using pulmonary function tests, such as forced expiratory volume measurement (FEV1). Patients with an estimated FEV1 <50% are classified as serious. Pulmonary function is closely linked to mortality, since only about 5% of COPD patients with severe COPD die within 12 years of age, compared with only 5% of mild to moderate patients. COPD is the fourth leading cause of death worldwide (World Health Organization (WHO), World Health Report, Geneva, 2000. URL: http://www.who.int/whr/2000/en/whr00_annex_en.pdf (Lopez AD, Shibuya K, Rao C et al., 2006), chronic obstructive pulmonary disease: current burden and future projections, Eur Respir J, 27 (2), 397-412). Worsening is a key factor in a vicious circle of illness and is largely responsible for most COPD admissions (BTS (British Thoracic Society), 2006, Burden of Lung Disease Report, 2 ^nd ed, http://www.brit-thoracic.org .uk / Portals / 0 / Library / BTS% 20Publications / burden of_lung_disease2007.pdf). The average annual incidence was 2.3 for symptom-defined deterioration and 2.8 for health-defined deterioration (O'Reilly JF, Williams AE, Holt K et al., 2006, Prim Care Respir J. 15 (6): 346 -53). Early diagnosis and improved management and improved control of patient deterioration will help reduce the burden on resources already burdened by these hospitalizations. Available therapies for COPD are primarily temporary and there is no available therapy to halt the progressive destruction of airways or the reduction of pulmonary function associated with the disease. Current therapeutic agents, such as short- and long-lasting [beta] -adrenergic bronchodilators, inhaled anticholinergics (muscarinic antagonists) and inhaled corticosteroids are used to treat the symptoms and worsening of the disease. The main limitation of current corticosteroid regimens is that they are ineffective because they show resistance to corticosteroids and inactivate the anti-inflammatory action of these drugs. Clearly, there remains a large unmet medical need for new drugs to prevent progression of COPD. Chemokine receptor antagonists are an attractive approach to COPD therapy because inflammatory-cells that are exchanged in COPD are modulated by multiple chemokines, and thus blockade of chemokine receptors using LMW antagonists can be an effective anti-inflammatory strategy for the disease. An important feature of COPD is the amplification of the inflammatory response seen in normal smokers, so the goal of therapy is not to completely inhibit inflammatory cell infiltration but to lower it to the level observed in normal smokers without COPD. By acting specifically, anti-CXCR2 will prevent general immunosuppression associated with steroids - conservation of CXCR1 activity will allow baseline neutrophil activation that is important for host defense of COPD and CF. Although most COPD drugs are currently administered by inhalation to reduce systemic side effects, systemic administration will be optimal since chemokine antagonists act on receptors expressed in circulating inflammatory cells. This will provide an efficient method for reaching invasive micropulmonary and pulmonary parenchyma in COPD.

Unlike cytokines and interleukin receptors, chemokine receptors belong to the 'drug-targetable' superfamily of 7TM-GPCRs. Nevertheless, early attempts to discover potent antagonists faced more difficulties than anticipated based on experiments using GPCRs with small peptides or biogenic amine ligands. Efforts to small molecule drug-discovery programs focused on chemokine-receptor antagonists have gradually begun to understand the traits of chemokine receptors and the structural elements required for small molecules to act as antagonists. Interestingly, the structural diversity of CC-chemokine-receptor antagonists, marked by a number of essentially distinct series of chemicals identified, is significantly higher than that of CXC-chemokine-receptor antagonists, Suggesting that there may be differences between the two classes.

Chemokine receptors have generally proved to be difficult targets to antagonize, and great effort was needed to identify potent and selective CXCR2 antagonists. A first low molecular weight CXCR2 antagonist was described in 1998, and since then many non-competitive allosteric CXCR2 antagonists have been developed, some of which are undergoing clinical trials. Nevertheless, there is clearly a need for better and more potent antagonists of CXCR2 function.

The molecule of the immunoglobulin class has greatly increased its clinical utility over the past decade. Its specificity to the target and its ability to manipulate it using recombinant techniques provide tremendous potential for developing highly induced therapeutic therapies for the disease. Many types of immunoglobulin molecules and modified immunoglobulin molecules, including conventional four-chain antibodies, Fab and F (ab) 2 fragments, single domain antibodies (D (ab)), single chain Fvs and Nanobodies, And is potentially available to be manipulated. These will be further discussed herein in connection with the present invention relating to polypeptides constructed to act on at least two epitopes of CXCR2.

It is therefore an object of the present invention to provide a novel means for the prophylaxis or treatment of chronic obstructive pulmonary disease or COPD and other diseases associated with abnormal function of chemokine receptor CXCR2.

It is a further object of the present invention to provide a means of treating or preventing COPD and other diseases associated with abnormal function of CXCR2, which is an immunotherapy.

Yet another object of the present invention is to provide a polypeptide comprising an immunoglobulin CDR that is an antagonist of CXCR2 signaling.

Summary of the Invention

The invention relates to a polypeptide comprising at least two immunoglobulin antigen binding domains, wherein said polypeptide acts on or binds to a chemokine receptor CXCR2, said polypeptide comprising a first antigen binding domain which recognizes a first epitope on CXCR2 and And a second antigen binding domain that recognizes a second epitope on CXCR2. Preferred polypeptides of the present invention comprise a first antigen binding domain capable of binding to a linear peptide consisting of the sequence of amino acids set forth in SEQ ID NO: 7 and a second antigen binding domain capable of binding less or less affinity to said linear peptide . Sequence 7 is the first 19 N-terminal amino acids of human CXCR2. A preferred polypeptide of the present invention is biparatopic. As used herein, the term "double-paratovenous" means that the polypeptide comprises two antigen binding domains that recognize two different epitopes on the same protein target. However, multiple, or multiple, paratope and multivalent, i. E., Polypeptides that also have an antigen binding domain that recognizes one or more other target proteins, recognize multiple, paratope, i. E., Three or more epitopes on the same target protein Are also included within the scope of the present invention.

In a preferred polypeptide of the invention, the amino acid sequence comprising the amino acid sequence comprising the first antigen binding domain and the second antigen binding domain is linked by a linker region. As discussed in more detail herein, the linker is preferably a peptide, although it may or may not originate from an immunoglobulin.

In a particularly preferred polypeptide according to the present invention said first antigen binding domain is comprised within a first immunoglobulin single variable domain and said second antigen binding domain is comprised within a second immunoglobulin single variable domain. At least one of the first and second immunoglobulin single variable domains may be a V _L domain or a fragment thereof or may be a V _H domain or a fragment thereof. A polypeptide in which each of the first and second antigen binding domains is contained in a V _L domain or a fragment thereof, or wherein each of the first and second antigen binding domains is contained in a V _H domain or a fragment thereof is encompassed by the present invention. The polypeptides of the present invention may include both V _L and V _H amino acid sequences or fragments thereof in a single molecule.

In a further preferred embodiment, the first and second antigen binding domains are comprised within first and second immunoglobulin single variable domains which are domain antibodies (dAbs). In a most preferred embodiment, at least one and preferably both of said first and second antigen binding domains are derived from a camelid or a humanized version thereof in which at least one humanized substitution is introduced into framework regions Is comprised within the immunoglobulin single variable domain which is a V _HH domain from a single heavy chain of heavy chain antibodies or a fragment thereof.

The immunoglobulin single variable domain with a V _HH sequence of amino acids from a heavy chain monoclonal antibody of the kind obtainable from camelid or a fragment or variant thereof is referred to herein as "V _HH domain" or a fragment or " Can be mentioned. Nanobodies®, Nanobodies® and Nanoclone® are available from Ablingen NV. (Ablynx NV).

In the polypeptides of the present invention, each antigen binding domain comprises at least one CDR, preferably two or three CDRs, as defined herein. In preferred polypeptides of the invention, the preferred structure of the immunoglobulin single variable domain is of a V _HH domain or nano-body having the following structure:

FR-CDR-FR-CDR-FR-CDR-FR

Here, CDRs and FRs are further defined herein.

Preferred dual parathetic nanobodies according to the present invention have one of the following structures:

i) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 linker-HLE

iii) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-HLE-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 contains the second antigen domain (the first antigen binding domain) when FR1-CDR1-FR2-CDR2-FR3-CDR3- FR6-CDR5-FR7-CDR4-FR7-CDR3-CDR3-FR4-CDR3-FR4-CDR3-FR4-CDR3-FR4- -CDR6-FR8 comprises a first antigen binding domain (linear sequence 7 binding agent), and HLE is a binding unit that provides an increased in vivo half-life.

Such preferred fragments or variants of the double-paratope nano-bodies are those in which the CDRs and FRs originate from the camelid, or embodiments in which one of the FRs has at least one humanized substitution and is preferably fully humanized. Are included in the invention.

Particularly preferred dual parathetic nanobodies according to the present invention are those designated herein as 163D2 / 127D1, 163E3 / 127D1, 163E2 / 54B12, 2B2 / 163E3, 2B2 / 163D2, 97A9 / 2B2 and 97A9 / 54B12 Its amino acid sequence is set forth in Table 13), and in particular its variants having the sequence optimized amino acid substitutions as defined herein, such as those set forth in Table 32 for the component nano-bodies.

Additional particularly preferred dual parathetic nanobodies according to the present invention are those shown in Table 33. &lt; tb &gt; &lt; TABLE &gt;

The polypeptides of the present invention are modulators of CXCR2 signaling and block, reduce or inhibit CXCR2 activity. They can inhibit the binding of natural ligands, e. G., Gro-a, to human CXCR2 to an IC50 of less than 20 nM. Preferably, the polypeptides of the present invention and especially the dual paratovenous nanobodies of the present invention are selected from the group consisting of the 163D2 / 127D1, 163E3 / 127D1, 163E3 / 54B12, 163D2 / 54B12, 2B2 / 163E3, 2B2 / 163D2, 97A9 / RTI ID = 0.0 &gt; 97A9 / 54B12. &Lt; / RTI &gt;

The present invention described herein includes monovalent polypeptide building blocks used in the construction of double or multiple paratope or multivalent nanobodies. Preferred monovalent nanobodies are each and every polypeptide having an amino acid sequence as shown in Table 9, Table 34, or at least one framework region having an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in Table 9 or Table 34 . Preferred monovalent nanobodies are those shown in Table 9, but with at least one sequence optimization, substitution in the framework region, such as the nanobodies set forth in Table 32 or Table 34. Particularly preferred are monovalent nanobodies designated 137B7 and their sequence-optimized versions.

Preferred polypeptides of the invention bind to an epitope comprising the amino acids F11, F14 and W15 of SEQ ID NO: 1 (CXCR2). In a preferred dual paratopoietic polypeptide of the invention, such as a double-paratovenous nanobody, the second antigen binding domain comprises an epitope (amino acid residues 106-120, 184-208 and 274-294 of SEQ ID NO: 1) in the outer loop of human CXCR2, Lt; / RTI &gt; In one embodiment of the invention, the epitope has a stereostructure. In one embodiment of the invention, the epitope comprises amino acid residues W112, G115, I282 and T285 of SEQ ID NO: 1.

The invention also encompasses nucleic acid molecules encoding any polypeptide according to the invention and nucleic acids encoding the fragments thereof, such as nucleic acids encoding individual nanobodies comprised in a double-paratovenous nanobody. Also included in the present invention are vectors comprising the nucleic acids of the invention and host cells comprising said vectors and capable of expressing the polypeptides according to the invention.

In another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide according to the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient. The polypeptides of the present invention are useful for the treatment of diseases in which abnormal signal transduction from CXCR2 plays a role because they can block, inhibit or reduce the activity of CXCR2. The disease is selected from the group consisting of atherosclerosis, glomerulonephritis, inflammatory bowel disease (Crohn's disease), angiogenesis, multiple sclerosis, psoriasis, age-related macular degenerative diseases, Behcet's disease, uveitis, non- , Pancreatic cancer, esophageal cancer, melanoma, hepatocellular carcinoma, or ischemic perfusion injury. The disease may also be selected from the group consisting of respiratory tract conditions such as cystic fibrosis, severe asthma, aggravated asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, airway remodeling, obstructive bronchiolitis syndrome, Dysplasia. &Lt; / RTI &gt;

In a particularly preferred embodiment, the polypeptides of the present invention are useful in the treatment of worsening of COPD or COPD characterized by migration of leukocytes, particularly neutrophils to the lung parenchyma (mediated through CXCR2 signaling) and subsequent disruption thereof, &Lt; / RTI &gt; The polypeptides of the present invention are good candidates for use in the prevention or treatment of such diseases due to their ability to block, inhibit or reduce CXCR2 activity.

For human therapy, it is preferred that the polypeptides of the invention act on or specifically bind to human CXCR2. However, in order to be able to carry out a suitable toxicity test in a monkey, it is preferred that said polypeptide is capable of cross-reacting with primate CXCR2, particularly Cynomolgus monkey CXCR2. The polypeptides of the present invention may act or specifically bind to CXCR2 homologues from other species when the use of the water is considered.

Other aspects of the invention will become apparent from the further discussion herein.

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In the description, examples and claims herein,

a) Unless otherwise indicated or specified, all terms used will have their ordinary meaning in the art and will be apparent to those skilled in the art. See, for example, the standard guide mentioned below (Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols 1-3, Cold Spring Harbor Laboratory Press (1989); Genes II, John Wiley &amp; Sons, New York, NY, (1987); Lewin, "Genes II ", John Wiley &amp; Sons, (1985)]; [Roitt et al., "Immunology" (1985)); [Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering ", 2nd edition, University of California Press, Berkeley, CA 6th Ed.), Mosby / Elsevier, Edinburgh (2001)] and Roight's Essential Immunology, 10 Ed. Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005)).

b) Unless otherwise indicated, the term " immunoglobulin "or" immunoglobulin sequence "refers to a full-length antibody, its individual chains, and all Domain, or fragments thereof (each including an antigen-binding domain or fragment, such as, but not limited to, a V _HH domain or a V _H / V _L domain). Also, as used herein, the term "sequence" (eg, in the terms "immunoglobulin sequence", "antibody sequence", "variable domain sequence", " _VHH sequence" or "protein sequence" Unless a more limited interpretation is required, it should generally be understood to include both the related amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding it.

c) Unless otherwise indicated, the term "immunoglobulin single variable domain" is used as a generic term including, but not limited to, an antigen-binding domain or fragment, respectively, such as a V _HH domain or a V _H or V _L domain. The term antigen-binding molecule or antigen-binding protein is used interchangeably and also includes the term nano-body. The immunoglobulin single variable domain may further comprise a light chain variable domain sequence (e.g., a V _L -sequence), or a heavy chain variable domain sequence (e.g., a V _H sequence); More specifically, it may be a heavy chain variable domain sequence derived from a conventional four chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. Thus, the immunoglobulin single variable domain may be an immunoglobulin sequence suitable for use as a domain antibody, or a domain antibody, a single domain antibody, or an immunoglobulin sequence suitable for use as a single domain antibody, an " dAb " , Or a _VH sequence, but not limited thereto. The invention encompasses immunoglobulin sequences of different origin, including mouse, rat, rabbit, donkey, human and camelike immunoglobulin sequences. Immunoglobulin single variable domains include full human, humanized, otherwise sequence optimized or chimeric immunoglobulin sequences. The structure of an immunoglobulin single variable domain and an immunoglobulin single variable domain is known in the art and herein as "Framework Region 1" or "FR1 &quot;; To "Framework Area 2" or "FR2 &quot;; To "framework area 3" or "FR3 &quot;; And " FR &quot;, respectively referred to as a framework region 4 "or" FR4 &quot;, respectively, Three complementarity determining regions or "CDRs " referred to as " region 1" or "CDR1 &quot;;" complementarity determining region 2 "

d) Unless otherwise indicated, all methods, steps, techniques and manipulations not specifically described in detail can be performed or performed in a manner known per se, which will be apparent to those skilled in the art. For example, the standard guide and the general background referred to herein and the additional references cited herein; And the following references describing, for example, protein manipulation techniques, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins: Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56); [Levin and Weiss, Mol. Biosyst. 2006, 2 (1): 49-57); [Irving et al., J. Immunol. Methods, 2001, 248 (1-2), 31-45); [Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12]; [Gonzales et al., Tumor Biol., 2005, 26 (1), 31-43].

e) Amino acid residues will be indicated according to standard three-letter or one-letter amino acid codes.

f) To compare two or more nucleotide sequences, the ratio of "sequence identity" between the first nucleotide sequence and the second nucleotide sequence is the ratio of the nucleotide sequence of the first nucleotide sequence to the nucleotide sequence of the first nucleotide sequence [identical to the nucleotide at the corresponding position in the second nucleotide sequence Number of nucleotides in the first nucleotide sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of nucleotides in the second nucleotide sequence - compared to the first nucleotide sequence - Considered as a difference at a single nucleotide (position)); Or may be calculated or determined using suitable computer algorithms or techniques. The degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0 using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A. In general, in order to determine the "sequence identity" ratio between two nucleotide sequences according to the calculation methodology outlined herein, the nucleotide sequence with the maximum number of nucleotides is considered the " first "nucleotide sequence and the other nucleotide sequence Quot; second "nucleotide sequence.

g) In order to compare two or more amino acid sequences, the "sequence identity" ratio (also referred to herein as "amino acid identity") between the first amino acid sequence and the second amino acid sequence is [relative position within the second amino acid sequence By dividing by [the number of amino acid residues in the same first amino acid sequence in the first amino acid sequence] divided by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%] (wherein each deletion of the amino acid residues in the second amino acid sequence, Insertion, substitution or addition - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. the "amino acid difference" defined herein; Or may be calculated or determined using suitable computer algorithms or techniques. In order to determine the "sequence identity" ratio between two amino acid sequences in accordance with the calculation methodology outlined herein, the amino acid sequence with the maximum number of amino acid residues is considered as the " first "amino acid sequence, Quot; second "amino acid sequence.

Also, when determining the degree of sequence identity between two amino acid sequences, one of ordinary skill in the art may consider so-called "conservative" amino acid substitutions as described in v) below.

Any amino acid substitutions applied to the polypeptides described herein can be made by analyzing the frequency of amino acid variations between homologous proteins of different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978 , Chou and Fasman, Biochemistry 13: 211, 1974, and Adv. Enzymol., 47: 45-149, 1978, and in Eisenberg et al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984; [Kyte &Doolittle; J Molec. Biol. 157: 105-132, 1981; and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986), all of which are incorporated herein by reference in their entirety. For the primary and secondary structures of the nanobodies, the crystal structure of the V _HH domain from llama is described, for example, in Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); [Spinelli et al., Natural Structural Biology (1996); 3, 752-757; And Decanniere et al., Structure, Vol. 7, 4, 361 (1999).

h) When comparing two amino acid sequences, the term "amino acid difference" means insertion, deletion or substitution of a single amino acid residue on the position of the first sequence relative to the second sequence; It is understood that two amino acid sequences may contain one, two or more of these amino acid differences.

i) When a nucleotide sequence or an amino acid sequence respectively refers to "comprising" or "essentially consists of another nucleotide sequence or amino acid sequence", which is another nucleotide sequence or amino acid sequence, this means that the latter nucleotide sequence or amino acid sequence May refer to a nucleotide sequence or an amino acid sequence, respectively, which has been previously introduced into the aforementioned nucleotide sequence or amino acid sequence, but more generally this means that in general the nucleotide sequence or amino acid sequence referred to earlier is in its sequence, , It is meant to include a stretch of a nucleotide or amino acid residue, respectively, having the same nucleotide sequence or amino acid sequence as the latter sequence, respectively. By way of non-limiting example, when the dual paratopoietic immunoglobulin single variable domain of the present invention, e. G., A nanobody, is referred to as comprising a CDR sequence, it is meant that the CDR sequence is a double-paratope nano- But more generally this means that generally the double-parathetic nanobodies of the present invention have within its sequence the same amino acid sequence as the CDR sequence, regardless of how the double-parathetic nanobodies are produced or obtained, &Lt; / RTI &gt; of the amino acid residues, respectively. It should also be noted that when the latter amino acid sequence has a specific biological or structural function, it preferably has essentially the same, similar or equivalent biological or structural function to the amino acid sequence referred to earlier (i. Amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, similar or equivalent biological or structural function. For example, when the dual parathetic nanobodies of the present invention are each referred to as comprising a CDR sequence or framework sequence, the CDR sequences and framework preferably comprise a CDR sequence or a CDR sequence in said double-paratope nano- And can function as a framework sequence. Also, when a nucleotide sequence is referred to as containing another nucleotide sequence, the first mentioned nucleotide sequence is preferably an amino acid sequence encoded by the latter nucleotide sequence when expressed in an expression product (e. G., A polypeptide) (I.e., the latter nucleotide sequence is present in the same decoding frame as the previously mentioned larger nucleotide sequence).

j) The nucleic acid sequence or amino acid sequence is generally at least one other component present in its source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule or at least one contaminant, Is regarded as "essentially isolated (in the form of)" when separated from its constituent, for example, compared to its natural biological source and / or reaction medium or culture medium from which the sequence is obtained. In particular, nucleic acid sequences or amino acid sequences are considered "essentially isolated " when purified to at least 2-fold, especially at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. The nucleic acid sequence or amino acid sequence present in "essentially isolated form" is preferably essentially homogeneous when determined using suitable techniques, such as using suitable chromatography techniques, such as polyacrylamide-gel electrophoresis.

k) The term "antigen binding domain" as used herein means an amino acid sequence in an immunoglobulin that contains at least one CDR and is in a stereogenic form recognizing a target antigenic determinant or epitope.

l) The terms "antigenic determinator" and "epitope" which may also be used interchangeably herein refer to amino acid sequences within the target CXCR2 that are recognized by the antigen binding domain whether in linear or non-linear conformations.

m polypeptides of the present invention which can (specifically) bind to and / or have affinity / specificity / specificity for a specific antigenic determinant, epitope, antigen or protein (or at least one portion, fragment or epitope thereof) , E. G., The double-paratovenous nanobodies or fragments thereof described herein are referred to as "acting against" or "against" the antigenic determinant, epitope, antigen or protein.

n) The term "specificity" refers to the number of different types of antigen or antigenic determinants to which a particular antigen-binding domain of a polypeptide of the invention can bind. The specificity of the antigen-binding protein for any particular antigen / epitope may also be determined by the method described in WO &lt; RTI ID = 0.0 &gt; 08/020079 &lt; / RTI &gt; (incorporated herein by reference), which describes some preferred techniques for determining the binding between a polypeptide and an associated antigen or epitope. Can be determined on the basis of affinity and / or avidity as described on page 56. In general, each antigen-binding proteins, each of the antigen binding domain from the (e. G. A polypeptide of the invention) are each independently selected from 10 ^-5 to 10 ^-12 moles / liter or less, preferably 10 ^-7 to 10 ^-12 moles / liters or less, more preferably 10 ^-8 to 10 ^-12 moles / liter of a dissociation constant (K _D) (i.e., 10 ⁵ to 10 ¹² liters / mole or more, preferably 10 ⁷ to 10 ¹² liters / mole or more, More preferably from 10 ⁸ to 10 ¹² liters / mole (K _A )) to the antigen / epitope thereof. Any K _D value greater than 10 ⁴ mol / liter (or any K _A value less than 10 ⁴ M ^-1 ) is generally considered to indicate non-specific binding. Preferably, the double-paratope polypeptides of the invention will bind to the required antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of the polypeptides of the present invention to CXCR2 can be determined, for example, by Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, Its different mutation methods known in the art; And other techniques referred to herein. &Lt; RTI ID = 0.0 &gt; The dissociation constant may be an actual or apparent dissociation constant, as is apparent to those skilled in the art and as described on pages 53-56 of WO 08/020079. Methods of determining the dissociation constants will be apparent to those skilled in the art and include, for example, the techniques mentioned in pages &lt; RTI ID = 0.0 &gt; 53-56 &lt; / RTI &

o) The half life of the polypeptides of the invention, in particular of the double-paratovenous nanobodies according to the invention, is generally such that the serum concentration of the polypeptide of the invention is lowered, for example by the degradation of the polypeptide and / or the elimination or removal of the polypeptide by its natural mechanism As a time taken to in vivo decrease by 50%. The in vivo half-life of the polypeptides of the present invention can be determined in any manner known per se, for example, by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art, for example, as described generally in paragraph o) of page 57 of WO 08/020079. Also, as mentioned in page 57 of WO 08/020079, half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta and area under the curve (AUC). See, for example, the experimental part below and a standard guide such as Kenneth, A. et al: A Handbook for Pharmacists and Peters et al., Pharmacokinete analysis: A Practical Approach (1996) . See also "Pharmacokinetics ", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The term " increased half-life "or" increased half-life "means an increase in t1 / 2-beta with or without an increase in t1 / 2- alpha and / or AUC.

p) In the context of the present invention, "blocking, reducing or inhibiting " the activity of CXCR2 when measured using an appropriate in vitro, cellular or in vivo assay is the same condition, but in the absence of the polypeptide of the invention, , Such as at least 10% or at least 25%, such as at least 50%, at least 60%, more preferably at least 5%, such as at least 10% or at least 25%, relative to the activity of CXCR2, , At least 70%, at least 80%, or 90% or more.

As will be apparent to those skilled in the art, inhibition also results in a decrease in the affinity, binding strength, specificity and / or selectivity of CXCR2 to one or more of its ligands or binding partners, and / Or a reduction in the susceptibility of CXCR2 to one or more conditions (e.g., pH, ionic strength, presence of co-factors, etc.) in the medium or environment in which CXCR2 is present. As will be apparent to those skilled in the art, this can again be determined in any suitable manner and / or using any suitable assay known per se, depending on the target or antigen of interest.

q) As used herein, "modulating" means modulating the CXCR2 &lt; RTI ID = 0.0 &gt; And / or competition with a natural ligand for binding to CXCR2, and / or to decrease or inhibit binding of CXCR2 to one of its ligands. In addition, the modulator can be used to alter the folding or stereostructure of, for example, CXCR2, or to alter its stereostructure (e.g., upon binding of a ligand) to its ability to associate with other (lower) It can be accompanied by a change in the quality of the product. In addition, modulating may involve, for example, resulting in a change in the ability of CXCR2 to transport another compound or to act as a channel for another compound (e.g., an ion).

The modulation, particularly inhibition or reduction, of CXCR2 activity by the polypeptides of the present invention, particularly the dual paratopoietic nanobodies of the invention, may be reversible or irreversible, but will generally be reversible for pharmaceutical and pharmacological purposes.

r) The polypeptides of the present invention have affinities that are at least 10-fold, such as at least 100-fold, preferably at least 1000-fold, and up to 10,000-fold or more affinity than the affinity binding to the second target or polypeptide Quot; specific for "CXCR2 as compared to a second target or antigen when bound to CXCR2, wherein the K _D value, K _A value, K _off rate and / or K _on rate are suitably expressed). For example, a polypeptide of the present invention may be at least 10-fold, such as at least 100-fold, preferably at least 1000-fold, such as 10,000-fold or even less than the K _D binding to another target or polypeptide or epitope thereof the value _D may be bonded to CXCR2.

s) The terms "cross-blocking", "cross-blocked" and "cross-blocking" refer to the ability of an immunoglobulin single variable domain or polypeptide to inhibit the binding of another immunoglobulin single variable domain or polypeptide of the invention to a given target Is used interchangeably herein to mean. The extent to which an immunoglobulin single variable domain or polypeptide of the present invention inhibits binding of another target and thus may be referred to as cross-blocking in accordance with the present invention may be determined using competitive binding assays. Particularly suitable quantitative cross-blocking assays are the use of labeled (e.g. His-tagged, radioactive or fluorescently labeled) immunoglobulin single variable domains or polypeptides according to the invention in combination with a binding partner with other binders Lt; / RTI &gt; or FACS- or ELISA-based methods to measure competition between the two. The experimental part generally describes suitable FACS- and ELISA-alternative-based assays to determine if binding molecules can cross-block or cross-block immunoglobulin single variable domains or polypeptides according to the invention. It will be appreciated that assays may be used with any of the immunoglobulin single variable domains or other binding agents described herein. Thus, in general, the cross-cutting amino acid sequence or other binding agent according to the invention is characterized in that the recorded substitution of an immunoglobulin single variable domain or polypeptide according to the invention in the presence of a second amino acid sequence or other binding agent of the invention during the assay, To 50% to 100% of the maximum theoretical substitution by a potential cross-linking agent (e. G., Another antibody fragment, V _HH , dAb or similar V _H / V _L variant).

t) If the polypeptide according to the invention is specific for both different antigens or antigenic determinants (as defined herein), two different antigens or antigenic determinants (such as two different species of mammal, Quot; cross-reactive "with respect to serum albumin or CXCR2 from cynomolgus monkeys).

u) Conservative amino acid changes, as defined herein, include amino acid substitutions where the amino acid residue is replaced by another amino acid residue of similar chemical structure and has little or essentially no effect on the function, activity or other biological properties of the polypeptide . Such conservative amino acid substitutions are known in the art, e.g. from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; The (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings from WO 04/037999 and WO 98/49185 and from further references cited herein.

The conservative substitution is preferably a substitution in which one amino acid in the following group (a) - (e) is replaced with another amino acid residue in the same group: (a) a small aliphatic, non-polar or low polarity residue: Ala, Ser , Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polarity, positive charge residue: His, Arg and Lys; (d) a large aliphatic, nonpolar residue: Met, Leu, Ile, Val and Cys; And (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Gly or Ser of Ala; Lys of Arg; Gln or His of Asn; Glu of Asp; Ser of Cys; Asn of Gln; Asp of Glu; Ala or Pro of Gly; Asn or Gln of His; Leu or Val of Ile; Ile or Val of Leu; Arg, Gln or Glu of Lys; Leu, Tyr or Ile of Met; Met, Leu or Tyr of Phe; Thr of Ser; Ser of Thr; Tyr of Trp; Trp of Tyr; And / or substitution of Phe with Val, Ile or Leu.

v) As used herein, a CDR is a complementarity determining region of a polypeptide of the invention. A CDR is a stretch of an amino acid that alone or in combination with one or more other CDRs establishes complementarity with the antigen (s) or epitope (s) recognized by the polypeptide of the present invention. CDRs are identified in the amino acid sequence by a specific numbering protocol. In the claims and the detailed description of the present application, Kabat numbering is used.

w) As used herein, FR is a framework region (sometimes called FW). A framework region is a stretch of amino acids that flank one or more CDRs and support it in the correct three-dimensional conformation for antigen or epitope recognition. FRs are not specific to the target antigen or epitope, but are specific to the species origin or species of the immunoglobulin molecule in which they are present. As discussed in detail herein, a range of amino acid sequences of framework regions that are engineered to be different from the source of immunoglobulins, e. G., The framework sequences applied by camelid, are present in the polypeptides of the present invention.

x) As used herein, CXCR2 refers to a cytokine receptor that is present on the surface of a leukocyte and to which the naturally occurring ligand can be a Gro-alpha, beta, gamma, IL-8, ENA-78 or GCP-2 . CXCR2 generally refers to any protein that exhibits CXCR2 function, regardless of the species from which it is derived. However, as used herein, human CXCR2 refers to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or any allelic variant or orthologue thereof, and Cynomolgus CXCR2 refers to the amino acid sequence set forth in SEQ ID NO: &Lt; / RTI &gt; or any of its allelic variants or orthologs.

y) As used herein, "sequence-optimized" means facilitating substitution, insertion or deletion in an amino acid sequence to ensure certain properties or structural characteristics that may not be present in the native sequence. Such substitution, insertion or deletion can be performed, for example, for chemical stabilization, improvement of manufacturing ability, formation of fatigue glutamate, or prevention of oxidation or isomerization. Methods for achieving optimization of the above properties, which may be used in the dual paratopoietic polypeptides of the present invention, particularly in the dual parathetic nanobodies, are described in WO 2009/095235 incorporated herein by reference. In addition, sequence optimization techniques can be performed to humanize the double paratope polypeptides of the invention in the manner described herein. Thus, when used herein, sequence optimization, sequence optimization, or sequence-optimized, this refers specifically to humanized substitutions or insertions, as well as to partially or fully humanized double-paratope polypeptides, preferably double-paratope nano-bodies .

DETAILED DESCRIPTION OF THE INVENTION

As described above, in a first aspect, the present invention provides a polypeptide comprising at least two immunoglobulin antigen binding domains, wherein said polypeptide acts on or binds to the chemokine receptor CXCR2, wherein said polypeptide comprises a CXCR2 1 epitope and a second antigen binding domain that recognizes a second epitope on CXCR2.

Preferred polypeptides of the present invention comprise a first antigen binding domain capable of binding to a linear peptide consisting of the sequence of amino acids set forth in SEQ ID NO: 7 and a second antigen binding domain capable of binding less or less affinity to said linear peptide . Sequence 7 is the first 19 N-terminal amino acids of human CXCR2.

In one embodiment, the first antigen binding domain recognizes a first epitope within or comprising amino acids 1 to 19 of CXCR2, and the second antigen binding domain recognizes a second epitope on CXCR2 outside of amino acids 1 to 19 .

The first and second antigen binding domains may be included in one or more amino acid sequences that are characterized by molecules of the immunoglobulin class. For example, the peptides or polypeptides include conventional four chain antibodies each linked by a linker. In particular, the polypeptide of the present invention is characterized in that said first and second antigen binding domains are respectively contained in first and second antibodies each comprising two heavy chains and two light chains, and said first and second antibodies are linked by a linker . Alternatively, the first and second antigen binding domains may be comprised within a single antibody comprising two heavy chains and two light chains.

In another embodiment of the invention, the first and second antigen binding domains are contained within an amino acid sequence that is a heavy chain antibody, and in particular the polypeptide of the invention is characterized in that said first antigen binding domain is contained within a first heavy chain antibody, Binding domain may be comprised within a second heavy chain antibody and the first and second heavy chain antibodies may be linked by a linker. The heavy chain antibody may be obtained from the camel family and may in fact comprise only two heavy chains, each with an immutable and variable region. Polypeptides wherein the first and second binding domains are included in a single heavy chain antibody comprising two heavy chains are also contemplated by the present invention.

Another embodiment of a peptide or polypeptide comprising first and second binding domains may be a single chain Fv (scFv). These include linear fusions of the V _L and V _H domains. Thus, the polypeptide of the present invention may be such that the first and second antigen binding domains are contained within first and second antibody single chain Fv (scFv) fragments, respectively, and the first and second scFv fragments are linked by a linker .

In a further embodiment, the first and second antigen binding domains can be included in one or more Fab or F (ab) 2 fragments of a conventional four chain antibody. Fab fragments comprise one constant domain and one variable domain from each of one heavy chain and one light chain of a conventional antibody. The F (ab) 2 fragment comprises two Fab fragments linked by a portion of the hinge region of a conventional antibody. In particular, the polypeptide of the present invention is characterized in that said first and second antigen binding domains are contained within first and second antibody Fab or F (ab) 2 fragments, respectively, and said first and second Fab or F (ab) It may be connected by a linker. In such an embodiment, the first antigen binding domain can be contained within an antibody Fab fragment and the second antigen binding domain can be contained within an F (ab) 2 fragment or vice versa.

In a preferred embodiment of the present invention, the first antigen binding domain is comprised within a first immunoglobulin single variable domain and the second antigen binding domain is comprised within a second immunoglobulin single variable domain.

A specific example of this embodiment of the invention is that the first and second antigen binding domains are comprised in a domain antibody called d (ab). d (ab) comprises a single V _L or V _H domain from a conventional antibody. Accordingly, the polypeptide of the present invention may be such that the first and second antigen binding domains are contained in the first and second domain antibodies (dAb), and the first and second dAbs are linked by a linker. The first and second dAbs may be antibody V _L fragments or antibody V _H fragments. In such embodiments, the first antigen binding domain can be included in the V _L fragment, and the second antigen binding domain can be included in the V _H fragment or vice versa.

For a general description of (single) domain antibodies, see also EP 0 368 684. (Holt et al., Trends Biotechnol., 2003, 21 (11)) for the term "dAb ", for example in Ward et al., (Nature 1989 Oct 12; 341 : 484-490]; And, for example, WO 06/030220, WO 06/003388 and Dormant. (Domantis Ltd.). It should also be noted that single domain antibodies or single variable domains may be derived from a particular species of shark (e.g., the so-called "IgNAR domain ", e.g., See WO 05/18629).

The single chain variable region of the heavy chain antibody is known as the V _HH domain and includes antibody fragments known as nanobodies. The nanobody may comprise the entire V _HH domain or fragment thereof. For a general description of heavy chain antibodies and their variable domains, see the prior art mentioned in WO08 / 020079 on page 59 and the list of references mentioned on pages 41 to 43 of the international patent application WO06 / 040153. The V _HH domain contains many unique (or both) functional domains that make the isolated V _HH domain (and _nanobodies based on it having the same structural and functional properties as the naturally occurring V _HH domain) and polypeptides containing it as highly functional antigen binding domains or polypeptides Structural and functional properties. In particular, the V _HH domain (designed " designed to functionally bind native antigen " in the absence of or without any interaction with the light chain variable domain) and nanobodies may be single, Domain or protein. As used herein, the term nanobody includes naturally occurring V _HH domains and fragments thereof, as well as variants and derivatives thereof discussed in detail herein.

In a most preferred embodiment of the invention, the dual paratoptic polypeptide of the invention is characterized in that said first antigen binding domain is comprised within a first nanobody and said second antigen binding domain is comprised within a second nanobody, And the second nano body is connected by the linker.

The structure of the V _HH domain is

FR-CDR-FR-CDR-FR-CDR-FR

And the double paratope polypeptides of the present invention may have one of the following structures:

i) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 linker-HLE

iii) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-HLE-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 contains the second antigen domain (the first antigen binding domain) when FR1-CDR1-FR2-CDR2-FR3-CDR3- FR6-CDR5-FR7-CDR4-FR7-CDR1-FR2-CDR3-FR4-CDR3-FR4-CDR3-FR4-CDR3- -CDR6-FR8 comprises a first antigen binding domain (linear sequence 7 binding agent), and HLE is a binding unit that provides an increased in vivo half-life.

Thus, "double paratope nano-bodies according to the present invention " as used herein means a polypeptide comprising two single nanobodies linked by a linker.

However, the dual parathetic nanobodies of the present invention may contain only one CDR in each nanobody. In that case, preferred CDRs are CDR3 and / or CDR6. However, the double-paratovenous nanobodies according to the present invention are capable of binding CDR1 or CDR2 or CDR3 or CDR1 and CDR2 or CDR1 and CDR3 or CDR2 and CDR3 or CDR1 and CDR2 and CDR3 to the N-terminal nano-body, May include any one of the following combinations: CDR4 or CDR5 or CDR6 or CDR4 and CDR5 or CDR4 and CDR6 or CDR5 and CDR6 or CDR4 and CDR5 and CDR6. As indicated above, the double-paratovenous nanobodies of the invention may comprise all CDR1, CDR2, CDR3, CDR4, CDR5 and CDR6, wherein each CDR is flanked by a FR.

The FRs may have an amino acid sequence consistent with the camelid source. However, in preferred embodiments, at least one FR has at least one sequence optimized amino acid substitution, preferably one or more, more preferably all FRs are partially or fully humanized. Substitutions for sequence optimization are discussed in more detail below.

Also included in the first and second immunoglobulin single variable domains are the first and second antigen binding domains, not the nanobodies, but the domains or fragments of conventional antibodies as discussed above, such as human antibodies, domains or fragments In this embodiment of the present invention, it is mentioned herein that the CDR (s) therein is changed to at least one camelised substitution, and optionally the production of fully camelised CDRs is possible.

As further described herein, the total number of amino acid residues in a single nano-body can be 110-120, preferably 112-115, and most preferably 113. However, parts, fragments, analogs or derivatives of the nanobodies (which are further described herein) are intended to encompass all such parts, fragments, analogs or derivatives which meet the additional requirements outlined herein and which are preferred for the purposes described herein Is not particularly limited to its length and / or size, if appropriate.

As further described herein, the amino acid residues of the nanobodies are described in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; (See, e.g., Kabat et al., ("Sequence of proteins of immunological interest &quot;) as applied to the V _HH domain from camelid ", US Public Health Services, NIH Bethesda, MD, Publication No. 91)] are numbered according to the general numbering for V _H domain set forth, according FR1 of Nanobodies may comprise an amino acid residue in position 1-30 , The CDR1 of the nanobody may comprise an amino acid residue at position 31-35, the FR2 of the nanobody may comprise an amino acid residue at position 36-49, and the CDR2 of the nanobody may contain an amino acid residue at position 50-65 The FR3 of the nanobody may comprise amino acid residues at positions 66-94, the nanobody CDR3 may comprise amino acid residues at positions 95-102, and the nanobody FR4 may comprise positions 103-113 Lt; / RTI &gt; amino acid residues. In the preferred dual parathetic nanobodies of the present invention, the N-terminal nano-body may have FRs and CDRs at the positions indicated above, and FR-5 of the nanobodies at the C-terminal nano-body may contain amino acid residues at positions 1-30 The CDR4 of the nano-body may comprise an amino acid residue at position 31-35, the nano-body FR6 may comprise an amino acid residue at position 36-49, and the amino acid at the CDR5 position 50-65 of the nano- And the FR7 of the nanobody may comprise amino acid residues at positions 66-94, the nanobody CDR6 may comprise amino acid residues at positions 95-102, and the FR8 of the nanobody may contain amino acid residues at position 103 -113 &lt; / RTI &gt; amino acid residues.

However, it will be appreciated that the antibodies, in particular the CDRs and FRs of the nanobodies, can be identified by a numbering system that is an alternative to carbat. These include Chothia, IMGT and AHo systems. Identification of the positions of the CDRs or FRs of any one of the amino acid sequences identified in Tables 9, 13, 19, 32, 33 and 34 according to these alternative numbering systems can be achieved by analysis of the sequences. For this, you can refer to the following website: http://www.biochem.ucl.ac.uk/~martin/ (コ テ ィ ア); http://imgt.cines.fr (IMGT) and http://www.bio.uzh.ch/antibody/index.html (AHo). Specifically, in the preferred dual parathetic nanobodies of the invention described herein, CDR 1, 2, 3, 4, 5 or 6 can be defined by one of these numbering systems as an alternative to Kabat, And will also be included within the scope of the present invention.

The kotoa CDR for some nanobodies according to the present invention is shown in Table 35.

The nanobodies are nano-bodies that have a high level of sequence homology to the so-called "V _H 3 class" (i.e., V _H 3 class such as DP-47, DP-51 or DP- ), And this nano body is preferable for the construction of the double parathetic nano body of the present invention. However, any type of Nanobodies acting against the CXCR2, and for example so-called "V _H 4 classes" (i.e., V _H 4 classes, for example the human germline DP-78 as described in WO 07/118670 It should be noted that nanobodies belonging to the Nanobodies with high levels of sequence homology to the sequences can be used in the construction of the double-paratovenous nanobodies of the present invention.

Linker molecules linking one or more peptides or polypeptides comprising the first and second antigen binding domains according to the present invention may or may not be of immunoglobulin origin. When the polypeptide of the present invention is a double-paratopoietic immunoglobulin single variable domain, for example a nano-body, the linker may comprise a C-terminus of one immunoglobulin single variable domain comprising an antigen binding domain, Terminal of another immunoglobulin single variable domain.

Spacers or linkers suitable for use in the double paratope polypeptides of the present invention to link together the first and second antigen binding domains together, particularly two nanobodies, will be apparent to those skilled in the art and will generally be understood as linking amino acid sequences May be any linker or spacer used in the art. Preferably, the linker or spacer is suitable for use in constructing the protein or polypeptide for which the pharmaceutical application is intended.

For example, the linker may be an amino acid sequence of a suitable amino acid sequence, particularly 1 to 50, preferably 1 to 30, such as 1 to 10 amino acid residues. A preferred example of a portion of the amino acid sequence is described in WO 99/42077, for example, type (gly ser _{x y) z} of the gly-ser linker such as (gly ₄ ser) _3, or (gly ser _{2 3) 3,} and the present GS30, GS15, GS9 and GS7 linkers (see, for example, WO 06/040153 and WO 06/122825) described in the Ablings application cited above, and hinge-like regions such as the hinge region of a naturally occurring heavy chain antibody or similar sequence (As described, for example, in WO 94/04678).

Some other possible linkers are poly-alanine (e.g. AAA), and linker GS30 (SEQ ID NO: 85 of WO 06/122825) and GS9 (SEQ ID NO: 84 of WO 06/122825).

Preferred linkers in accordance with the present invention comprise from 3 to 50, for example 3 to 9, 10 to 15, 16 to 20, 21 to 25, 26 to 35, 36 to 40, 41 to 45 or 46 to 50 amino acids Peptide linker. In one embodiment of the invention, the peptide linker is 35 amino acids in length. The linker may consist of only two different amino acids. As noted above, these may be glycine and serine. Alternatively, they can be proline and serine.

In some embodiments of the present invention, particularly the double-paratovenous nanobodies of the invention, the peptide linker consists of the following amino acid sequence:

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 220).

Other suitable linkers generally include those suitable for use in organic compounds or polymers, particularly proteins for pharmaceutical applications. For example, a poly (ethylene glycol) moiety has been used to link the antibody domain (see, for example, WO 04/081026).

Thus, in another aspect, the invention is directed to a molecule comprising at least two polypeptides, wherein said molecule acts on or binds to the chemokine receptor CXCR2, wherein said first polypeptide comprises a first immunoglobulin antigen binding domain Wherein the second polypeptide comprises a second immunoglobulin antigen binding domain and wherein the first and second antigen binding domains recognize first and second epitopes on CXCR2 and wherein the at least two polypeptides are linked to a non- Lt; / RTI &gt;

Preferably, in aspects of the invention, the first antigen binding domain is capable of binding to a linear peptide consisting of the sequence of amino acids set forth in SEQ ID NO: 7, wherein said second antigen binding domain is capable of binding to said linear peptide Friendly manner. Preferably, the first epitope comprises or is within amino acids 1 to 19 of CXCR2 and the second epitope is outside amino acids 1 to 19 of CXCR2.

Preferably, in this aspect of the invention, the first and second antigen binding domains are comprised within an immunoglobulin single variable domain, wherein said first and second immunoglobulin single variable domains are preferably selected from the group consisting of nanobodies, Is an arbitrary nanobody specifically described in Fig.

In all aspects of the invention described herein, the essential property of the linker is that it has a length and a conformation allowing the first and second antigen binding domains to bind to their respective epitopes on CXCR2.

The linker (s) used may also comprise one or more moieties for imparting one or more other favorable properties or functionality to the polypeptides of the invention and / or for the formation of derivatives and / or for attachment of functional groups, Which is described herein for a derivative of a parathetic nano-body). For example, a linker containing one or more charged amino acid residues (see Table A-2 on page 48 of International Patent Application WO 08/020079) may provide improved hydrophilic properties while a smaller epitope or tag Linkers that form or contain can be used for detection, identification and / or purification. Again, based on the disclosure herein, one of ordinary skill in the art will be able, after some limited conventional experimentation, to determine an optimal linker for use with a particular polypeptide of the invention.

Finally, when two or more linkers are used in the polypeptides of the present invention, these linkers may be the same or different. Again, based on the disclosure herein, one of ordinary skill in the art will be able, after some limited conventional experimentation, to determine an optimal linker for use with a particular polypeptide of the invention.

In general, for ease of expression and production, the polypeptide of the present invention will be a linear polypeptide. However, the present invention is not limited in its broadest sense. For example, if the polypeptide of the invention comprises three or more nanobodies, it is possible to link them using linkers with three or more "arms ", each" arm " Of the "nano-body ". Also, although less preferred, cyclic structures may be used. In particular, any alignment of two or more nanobodies using the identified one or more linkers may be prepared. For example, a double-paratope, bispecific nano-body comprising two immunoglobulin binding domains that act on or bind to CXCR2 and one or more immunoglobulin binding domains that act on or bind to human serum albumin (HSA) May be considered and the HSA binding domain may be included in a nanobody linked to a CXCR2 binding nano-body through a linker defined herein at any position, for example between two CXCR2 binding nano-bodies.

The present inventors have made the double paratope polypeptides according to the present invention. The amino acid sequences of the polyvalent and double-parathetic anti-CXCR2 nanobodies are set forth in Table 13 within the Examples herein. Of these, particularly preferred polypeptides according to the present invention are 163D2-127D1, 163E3-127D1, 163E3-54B12, 163D2-54B12, 2B2-163E3, 2B2-163D2, 97A9-2B2, 97A9-54B12, 127D1-163D2, 127D1-163E3 , 2B2-97A9, 54B12-163D2, 54B12-163E3, 163D2-2B2 and 163E3-2B2, and 127D1-97A9, 54B12-97A9 and 97A9-127D1, and their sequence-optimized variants designated in Table 13 . All of these double-parathetic nanobodies comprise a first nano-body comprising a first antigen binding domain capable of binding to a linear peptide consisting of the sequence of amino acids set forth in SEQ ID NO: 7 (amino acids 1-19 of CXCR2) And a second nano-binding domain comprising a second antigen-binding domain that binds with less affinity (see Table 8). Particularly preferred according to the invention are 163D2-127D1, 163E3-127D1, 163E3-54B12, 163D2-54B12, 2B2-163E3, 2B2-163D2, 97A9-2B2 and 97A9-54B12.

1) 163 D2 -127 D1 (SEQ ID NO: 58)

This embodiment of the present invention is characterized in that the second nanobody defined above has an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 145, Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141, 161 or 181 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably, the double-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 185, wherein said first nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 161, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 181 or comprises the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 145, 165, 185, 141,

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 145, 165, 185, 141, 161 or 181. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 104, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 124, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 120 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 83, 104, 124, 131, 79, 100, 85%, at least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: &Lt; / RTI &gt; polypeptides having sequence identity.

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 58, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering, Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual paratovogenic nanobodies of the invention comprise at least one sequence-optimized variant of SEQ ID NO: 58, but more than one sequence-optimized substitution, preferably a 163D2 nanobody, Substantially &lt; / RTI &gt; comprise a CDR sequence modified in the framework region to include one or more substitutions identified in Table 26 for the body as being suitable. Preferably, the 163D2 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 218 or SEQ ID NO: 218, and the 127D1 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 216. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

2) 163 E3 -127 D1 (SEQ ID NO: 59)

The embodiment of the invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 146, 166 or 186 Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141, 161 or 181 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably, the double-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 166, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 186, and said first nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 161, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 181 or comprises the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 Has at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 146, 166, 186, 141, 161 or 181.

Amino acid sequences may differ only in conservative amino acid changes from those set forth in any of SEQ ID NOs: 146, 166, 186, 141, 161 or 181. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 125, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 120 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 84, 105, 125, 131, 79, 100, , At least 85%, at least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a preferred embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid identity, or at least 85%, or at least 90%, or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: % Identity to the amino acid sequence of SEQ ID NO.

In one embodiment of this aspect of the invention, the double-paratope nano-body has at least 80% identity with the amino acid sequence or sequence 59 set forth in SEQ ID NO: 59 or at least 80% amino acid sequence identity with its framework region according to Kabat numbering , And the double-parathetic nanobodies can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another embodiment of this aspect of the invention, the dual parathetic nano-bodies of the invention comprise at least one sequence-optimized variant of SEQ ID NO: 59, but more than one sequence-optimized substitution, preferably a 163E3 nano- Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; Preferably, the 163E3 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217 and the 127D1 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 216. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

3) 163 E3 / 54B12 (SEQ ID NO: 62)

The embodiment of the invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 146, 166 or 186 Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171, and 191, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 151, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 166, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 186, and said first nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: , Preferably at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set out in SEQ ID NOs: 166, 186, 151, 171 or 191,

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 146, 166, 186, 151, 171, The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 125, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 Sequence.

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 84, 105, 125, 131, 89, 85%, or at least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a preferred embodiment, the double-paratope nano-body comprises an amino acid sequence having at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: do.

In one preferred embodiment, the dual parathetic nanobodies of the invention comprise at least 80% amino acid identity with the amino acid sequence or SEQ ID NO: 62, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering And the double paratope nano-body can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized substitution as set forth in SEQ ID NO: 62, but for at least one sequence-optimized substitution, preferably 163E3, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; Preferably, the 163E3 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217, and the 54B12 nano- , Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 219. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 212 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

4) 163 D2 / 54B12 (SEQ ID NO: 63)

The embodiment of the present invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 145, Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171, and 191, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 151, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 165, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 185, and said second nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: , Preferably at least 85%, or at least 90% or at least 95% amino acid identity with any one of the amino acid sequences set out in SEQ ID NOs: 165, 185, 151, 171,

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NOs: 145, 165, 185, 151, 171 or 191. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 104, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 124, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 Sequence.

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85% identical to the amino acid sequence shown in any of SEQ ID NOs: 83, 104, 124, 131, 89, 110, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a preferred embodiment, the dual paratope nano body of the invention comprises a polypeptide having at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: .

In one preferred embodiment, the dual parathetic nanobodies of the invention have at least 80% amino acid identity with the amino acid sequence or sequence 63 set forth in SEQ ID NO: 63, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 2.0E-09M.

In another embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized variant of SEQ ID NO: 63, but more than one sequence-optimized substitution, preferably a 163D2 nanobody, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; Preferably, the 163D2 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 218 or SEQ ID NO: 218, and the 54B12 nano- , Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 219. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

5) 2B2 / 163 E3 (SEQ ID NO: 64)

The embodiment of the present invention is characterized in that said first nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 147, 167 or 187 And the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 146, 166 or 186 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 167, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 187, and said second nano-body Wherein CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 166, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 186, or CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 comprises the amino acid sequence set forth in SEQ ID NO: At least 80%, preferably at least 85%, at least 90% or at least 95% amino acid identity with any one of the amino acid sequences set out in SEQ ID NOs: 167, 187, 164, 146 or 186. [

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NOS: 147, 167, 187, 146, 166 or 186. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to an aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 126, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 125 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 .

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85% identical to the amino acid sequence set forth in any of SEQ ID NOs: , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a preferred embodiment, the dual parathetic nanobodies according to the present invention have at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence shown in SEQ ID NO: 64 or the amino acid sequence of SEQ ID NO: &Lt; / RTI &gt;

Preferred embodiments of the double paratope nano-bodies of the present invention comprise an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 64 or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Sequence, and the double-parathetic nanobodies can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized variant of SEQ ID NO: 64, but more than one sequence-optimized substitution, preferably a 2B2 nanobody, Substantially includes the CDR sequences modified in the framework region to include one or more substitutions found to be appropriate in Table 24 for the body. Preferably, the 2B2 nano-body comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213 or 214 or SEQ ID NO: The body comprises an amino acid sequence having at least 80%, at least 85%, at least 90% or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

6) 2B2 / 163 D2 (SEQ ID NO: 65)

The embodiment of the present invention is characterized in that said first nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 147, 167 or 187 Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 145, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably, the double-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 167, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 187, and said second nano-body Wherein CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 185; or CDR1, CDR2, CDR3, CDR4, At least 80%, preferably at least 85%, at least 90% or at least 95% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NOS: 147, 167, 187, 145, 165 or 185.

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NOS: 147, 167, 187, 145, 165 or 185. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 126, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 104, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 124 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 Sequence.

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85%, at least 85% At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

Preferred dual paratope nano-bodies according to the present invention comprise a polypeptide having at least 80% amino acid identity, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 65 or SEQ ID NO: 65 .

Preferred embodiments of the double-paratope nano-bodies of the present invention comprise an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 65 or at least 80% amino acid sequence identity with its framework region according to Kabat numbering, Sequence, and the double-parathetic nanobodies can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise a double-paratope nano-body as set forth in SEQ ID NO: 65, but with one or more sequence-optimized substitutions, preferably 2B2 nanobodies, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 28 for the body as appropriate. Preferably, the 2B2 nano-body comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213 or 214 or SEQ ID NO: The body comprises an amino acid sequence having at least 80%, at least 85%, at least 90% or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 218 or SEQ ID NO: 218. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

7) 97A9 / 2B2 (SEQ ID NO: 47)

The embodiment of the present invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 143, 163 or 183 The first nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 147, 167 or 187 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 143, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 183 and the CDR4 The CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 147, the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 167, and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 187, or the CDR1, CDR2, CDR3, CDR4, At least 80%, preferably at least 85%, at least 90% or at least 95% amino acid identity with any one of the amino acid sequences set out in SEQ ID NOs: 163, 183, 147, 167,

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NO: 143, 163, 183, 147, 167 or 187. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to an aspect of the present invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 122, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 126 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 .

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85% identical to the amino acid sequence set forth in any of SEQ ID NOs: 81, 102, 122, 133, 85, 106, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

Preferred dual paratope nano-bodies include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 47 or the amino acid sequence set forth in SEQ ID NO:

In a particularly preferred embodiment, the dual paratope nano-body of the invention comprises an amino acid sequence having at least 80% amino acid identity with SEQ ID NO: 47 or at least 80% amino acid identity with its framework region according to Kabat numbering, Sequence, and the double-parathetic nanobodies can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized substitution as set forth in SEQ ID NO: 47, but with one or more sequence-optimized substitutions, preferably in Table 22 for &lt; Substantially &lt; / RTI &gt; include CDR sequences modified in framework regions to include one or more substitutions identified as appropriate in Table 20 for the &lt; RTI ID = 0.0 &gt; Preferably, the 97A9 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 215 or SEQ ID NO: 215 and the 2B2 nano- Or 214, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 213 or SEQ ID NO: 214. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

8) 97A9 / 54B12 (SEQ ID NO: 61)

The embodiment of the present invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 143, 163 or 183 Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171, and 191, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 151, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 143, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 183, and said first nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: At least 80%, preferably at least 85%, at least 90% or at least 95% amino acid identity with any one of the amino acid sequences set out in SEQ ID NOs: 143, 163, 183, 151, 171 or 191.

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NO: 143, 163, 183, 151, 271 or 191. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

According to this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 122, Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: 131 Sequence.

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 are at least 80%, at least 85% identical to the amino acid sequence shown in any of SEQ ID NOs: 81, 102, 122, 133, 89, 110, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a preferred embodiment, the double-paratope nano-body comprises an amino acid sequence as set forth in SEQ ID NO: 61 or a polypeptide having at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence of SEQ ID NO:

In a particularly preferred embodiment, the dual parathetic nanobodies of the present invention have at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 or at least 80% identity with its framework region according to Kabat numbering Amino acid, and the double-parathetic nanobody can inhibit the binding of Gro-? To human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the invention comprise at least one sequence-optimized variant of SEQ ID NO: 61, but more than one sequence-optimized substitution, preferably of 97A9 nano-bodies, Substantially includes the CDR sequence modified in the framework region to include one or more substitutions identified as appropriate in Table 30 for the body. Preferably, the 97A9 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence or SEQ ID NO: 215 shown in SEQ ID NO: 215 and the 54B12 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 219. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

9) 127 D1 / 163 D2 (SEQ ID NO: 53)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 141, Wherein the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165, and 185, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 145, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 161, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, and the CDR2 in the second nano- CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 185 or the amino acid sequence of CDR1, CDR2, CDR3, Or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 141, 161, 181, 145, 165 or 185. [

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 145, 165, 185, 141, 161 or 181. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 120, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR6 includes the amino acid sequence set forth in SEQ ID NO: 104, FR7 includes the amino acid sequence set forth in SEQ ID NO: 124 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80%, at least 85%, at least 80% At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobody comprises an amino acid sequence as set forth in SEQ ID NO: 53 or an amino acid sequence having at least 80% amino acid identity, at least 85%, at least 90% &Lt; / RTI &gt; polypeptides having sequence identity.

In another embodiment of this aspect of the invention, the double-paratope nano-body comprises an amino acid sequence as set forth in SEQ ID NO: 53 or an amino acid sequence having at least 80% amino acid sequence identity with the framework region of SEQ ID NO: And the double-parathetic nanobodies can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized variant of SEQ ID NO: 54, but for at least one sequence-optimized substitution, preferably 127D1 nanobodies, in Table 26 and 163D2 nano Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 28 for the body as appropriate. Preferably, the 127D1 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence or sequence 216 set forth in SEQ ID NO: 216 and the 163D2 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 218. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

10) 127 D1 / 163 E3 (SEQ ID NO: 54)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 141, Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence SEQ ID NO: 146, 166 or 186 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 161, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, and said second nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 166, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 186 or comprises the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 146, 166, 186, 141, 161 or 181.

The amino acid sequence may differ only in conservative amino acid changes from those shown in SEQ ID NO: 146, 166, 186, 141, 161 or 181. [ The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 120, 131, FR5 comprises the amino acid sequence shown in SEQ ID NO: 84, FR6 includes the amino acid sequence shown in SEQ ID NO: 105, FR7 includes the amino acid sequence shown in SEQ ID NO: 125 and / or FR8 includes the amino acid sequence shown in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80%, at least 85%, at least 80%, at least 80% At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 54 or the amino acid sequence set forth in SEQ ID NO: Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-paratope nano-body comprises at least 80% amino acid sequence identity with the amino acid sequence or SEQ ID NO: 54, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized substitution as set forth in SEQ ID NO: 54, but for at least one sequence-optimized substitution, preferably 127D1 nanobodies, Substantially includes the CDR sequences modified in the framework region to include one or more substitutions found to be appropriate in Table 24 for the body. Preferably, the 127D1 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence or sequence 216 set forth in SEQ ID NO: 216 and the 163E3 nanobody comprises SEQ ID NO: 217 Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 217. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

11) 127 D1 / 97A9 (SEQ ID NOS: 37 and 39)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 141, Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 143, 163 or 183 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 161, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, and the CDR2 in the second nano- CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 143, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 163, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 183 or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 141, 161, 181, 143, 163 or 183.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOS: 141, 161, 181, 143, 163 or 183. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 120, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 122 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80%, at least 85 , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-paratope nano-body comprises a first antigen binding domain comprising the amino acid sequence set forth in SEQ ID NO: 37 and a second antigen binding domain comprising the amino acid sequence set forth in SEQ ID NO: 37, and 39, having at least 80% amino acid identity, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NOS:

In another embodiment of this aspect of the invention, preferred embodiments of the double-parathetic nanobodies comprise the amino acid sequence shown in SEQ ID NOs: 37 and 39 or SEQ ID NOs: 37 and 39 and / or its framework region according to Kabat numbering at least 80 % Amino acid sequence identity, and said double-paratope polypeptide can inhibit the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one of the sequences shown in SEQ ID NOS: 37 and 39, but for at least one sequence-optimized substitution, preferably for the 127D1 nanobodies, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 22 for the 97A9 nanobodies. Preferably, the 127D1 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence or sequence 216 set forth in SEQ ID NO: 216 and the 97A9 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 215. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

12) 2B2 / 97A9 (SEQ ID NO: 46)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 147, Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 143, 163 or 183 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 167, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 187, and said second nano-body comprises CDR4 CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 143, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 163, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 183 or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 147, 167, 187, 143, 163 or 183.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOS: 147, 167, 187, 143, 163 or 183. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 126, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 122 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 are at least 80%, at least 85% identical to the amino acid sequence set forth in any of SEQ ID NOs: , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 46 or the amino acid sequence set forth in SEQ ID NO: Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 46, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering, Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized variant of SEQ ID NO: 46, but more than one sequence-optimized substitution, preferably a 2B2 nanobody, Substantially &lt; / RTI &gt; comprise a CDR sequence modified in the framework region to include one or more substitutions identified in Table 22 for the body as being suitable. Preferably, the 2B2 nano-body comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213 or 214 or SEQ ID NO: The body comprises an amino acid sequence having at least 80%, at least 85%, at least 90% or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 or SEQ ID NO: 215. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

13) 54B12 / 163 D2 (SEQ ID NO: 69)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171 and 191 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 151, Wherein the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165, and 185, or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 145, &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 191, and said second nano-body comprises CDR4 CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 185 or the amino acid sequence of CDR1, CDR2, CDR3, Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 151, 171, 191, 145,

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOS: 151, 171, 191, 145, 165 or 185. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 130, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR6 includes the amino acid sequence set forth in SEQ ID NO: 104, FR7 includes the amino acid sequence set forth in SEQ ID NO: 124 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80%, at least 85%, at least 80% At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 69 or SEQ ID NO: 69 Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence or sequence 69 set forth in SEQ ID NO: 69 or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the double-paratovenic nanobodies of the invention comprise at least one sequence-optimized variant of SEQ ID NO: 69, but for at least one sequence-optimized substitution, preferably for 54B12 nanobodies, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 28 for the body as appropriate. Preferably, the 54B12 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 219 or SEQ ID NO: 219, and the 163D2 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 218. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

14) 54B12 / 163 E3 (SEQ ID NO: 68)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171 and 191 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 151, Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 146, 166 or 186 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 191, and said second nano-body comprises CDR4 CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 166, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 186, or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 151, 171, 191, 146, 166 or 186.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOS: 151, 171, 191, 146, 166 or 186. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 130, 131, FR5 comprises the amino acid sequence shown in SEQ ID NO: 84, FR6 includes the amino acid sequence shown in SEQ ID NO: 105, FR7 includes the amino acid sequence shown in SEQ ID NO: 125 and / or FR8 includes the amino acid sequence shown in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another aspect, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80%, at least 85%, at least 80% At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 68 or the amino acid sequence set forth in SEQ ID NO: Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-paratope nano-body comprises at least 80% amino acid sequence identity with the amino acid sequence or sequence 68 set forth in SEQ ID NO: 68 or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized variant of SEQ ID NO: 68, but more than one sequence-optimized substitution, preferably of 54B12, Substantially includes the CDR sequences modified in the framework region to include one or more substitutions found to be appropriate in Table 24 for the body. Preferably, the 54B12 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 219 or SEQ ID NO: 219 and the 163E3 nano- Or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 217. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

15) 54B12 / 97A9 (SEQ ID NOS: 90 and 39)

The embodiment of the present invention is characterized in that the first nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 141, Wherein the second nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 143, 163 or 183 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 161, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, and the CDR2 in the second nano- CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 143, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 163, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 183 or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 141, 161, 181, 143, 163 or 183.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOS: 141, 161, 181, 143, 163 or 183. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 130, 131, FR5 comprises the amino acid sequence shown in SEQ ID NO: 81, FR6 comprises the amino acid sequence shown in SEQ ID NO: 102, FR7 contains the amino acid sequence shown in SEQ ID NO: 122 and / or FR8 contains the amino acid sequence shown in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85% identical to the amino acid sequence shown in any of SEQ ID NOs: 89, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 90 and 39, % &Lt; / RTI &gt; amino acid identity.

In another embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NOS: 90 and 39 or SEQ ID NOS: 90 and 39, or framework regions thereof according to Kabat numbering Wherein the double-paratope nano-body comprises an amino acid sequence having at least 80% amino acid sequence identity, wherein the double-paratope nano-body is capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one of the sequences shown in SEQ ID NOS: 90 and 39, but for one or more sequence-optimized substitutions, preferably for the 54B12 nano-bodies, Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 22 for the 97A9 nanobodies. Preferably, the 54B12 nano-body comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOS: 90 and 39, The 97A9 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 or SEQ ID NO: For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

16) 97A9 / 127 D1 (SEQ ID NOS: 39 and 37)

The embodiment of the present invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence SEQ ID NO: 143, 163 or 183 Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 161 and 181 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141, 161 or 181 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 143, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 183 and the CDR4 CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 161, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 181 or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 143, 163, 183, 141, 161 or 181.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 143, 163, 183, 141, 161 or 181. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 122, 133, FR5 comprises the amino acid sequence shown in SEQ ID NO: 79, FR6 comprises the amino acid sequence shown in SEQ ID NO: 100, FR7 includes the amino acid sequence shown in SEQ ID NO: 120 and / or FR8 includes the amino acid sequence shown in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80%, at least 85%, 80% , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90% or at least 70% of the amino acid sequence set forth in SEQ ID NOS: 39 and 37, And at least 95% amino acid identity.

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NOS: 39 and 37, or SEQ ID NO: 39 and / or 37, or a framework thereof according to Kabat numbering Region, wherein said double-paratope nano-body is capable of inhibiting the binding of &lt; RTI ID = 0.0 &gt; Gro-a &lt; / RTI &gt; to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one of the sequences shown in SEQ ID NOS: 39 and 37, but with one or more sequence-optimized substitutions, preferably in Table 22 for the 97A9 nano- Lt; RTI ID = 0.0 &gt; CDR &lt; / RTI &gt; sequence in the framework region to include one or more substitutions identified in Table 26 for 97A9 nanobodies. Preferably, the 97A9 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NOs: 39 and 37 or SEQ ID NOs: 39 and 37, The body comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence or sequence 216 set forth in SEQ ID NO: 216. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

17) 163 D2 / 2B2 (SEQ ID NO: 67)

The embodiment of the present invention is characterized in that the second nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 145, The first nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 147, 167 or 187 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 145, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 165, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 185, and the CDR4 CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 167, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 187, or wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, Or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 145, 165, 185, 147, 167 or 187. [

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 145, 165, 185, 147, 167 or 187. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 104, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 124, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 126 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 are at least 80%, at least 85% identical to the amino acid sequence set forth in any of SEQ ID NOS: 83, 104, 124, 131, 85, 106, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 67 or SEQ ID NO: Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence or sequence 67 set forth in SEQ ID NO: 67, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized substitution as set forth in SEQ ID NO: 67, but for one or more sequence-optimized substitutions, preferably for 163D2 nanobodies, Substantially &lt; / RTI &gt; include CDR sequences modified in framework regions to include one or more substitutions identified as appropriate in Table 20 for the &lt; RTI ID = 0.0 &gt; Preferably, the 163D2 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 218 or SEQ ID NO: 218, and the 2B2 nano- Or 214, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: 213 or SEQ ID NO: 214. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

18) 163 E3 / 2B2 (SEQ ID NO: 66)

The embodiment of the present invention is characterized in that the second nanobody defined above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 146, Wherein the first nano-body comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 147, 167 or 187 &Lt; / RTI &gt; wherein the at least one CDR comprises at least one CDR comprising a sequence.

Preferably the bi-parathetic nanobodies comprise the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 166, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 186, and said first nano-body CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 167, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 187 or comprises the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 Has at least 80% amino acid sequence identity, or at least 85% or at least 90% or at least 95% amino acid sequence identity with any one of the amino acid sequences set forth in SEQ ID NO: 146, 166, 186, 147, 167 or 187.

The amino acid sequence may differ only in conservative amino acid changes from those shown in any of SEQ ID NOs: 146, 166, 186, 147, 167 or 187. The amino acid sequence may differ from the sequence of any of the above sequence numbers only by 1, 2 or 3 amino acids.

In a preferred embodiment of this aspect of the invention, FR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 125, 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 126 and / or FR8 comprises the amino acid sequence set forth in SEQ ID NO: Lt; RTI ID = 0.0 &gt; amino acid &lt; / RTI &gt;

In another embodiment, FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 are at least 80%, at least 85% identical to the amino acid sequence set forth in any of SEQ ID NOs: 84, 105, 125, 131, 85, 106, , At least 90%, or at least 95% amino acid sequence identity.

For example, in this aspect of the invention, FR1 and / or FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 70 to 89, and FR2 and / or FR5 may comprise any one of SEQ ID NOS: FR3 and / or FR6 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 111 to 130, and FR4 and / or FR8 may comprise the amino acid sequence shown in any one of SEQ ID NOS: 131 to 133 Sequence.

In a particularly preferred embodiment of this aspect of the invention, the double-parathetic nanobodies comprise at least 80%, at least 85%, at least 90% or at least 95% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: Lt; / RTI &gt;

In another embodiment of this aspect of the invention, the double-parathetic nanobody comprises at least 80% amino acid sequence identity with the amino acid sequence or sequence 66 set forth in SEQ ID NO: 66, or at least 80% amino acid sequence identity with its framework region according to Kabat numbering Wherein said double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

In another preferred embodiment of this aspect of the invention, the dual parathetic nanobodies of the present invention comprise at least one sequence-optimized substitution as set forth in SEQ ID NO: 66, but for at least one sequence-optimized substitution, preferably for 163E3 nanobodies, Substantially &lt; / RTI &gt; include CDR sequences modified in framework regions to include one or more substitutions identified as appropriate in Table 20 for the &lt; RTI ID = 0.0 &gt; Preferably, the 163E3 nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217 and the 2B2 nano- Or 214, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NOS: 213 or 214, respectively. For example, the framework regions of these nanobodies may have sequences of framework regions FR1, FR2, FR3 or FR4 according to Kabat numbering, as shown in any one of SEQ ID NOS: 213 to 219 in Table 32. [ Preferably, the sequence-optimized double-parathetic nanobodies are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

As already discussed herein, the 163D2 / 127D1, 163E3 / 127D1, 163E3 / 54B12, 163D2 / 54B12, 2B2 / 163E3, 2B2 / 163D2, 97A9 / 2B2, 97A9 / 54B12, 127D1 / 163D2, 127D1 / 163E3, 127D1 / Preferred dual paratope nano-bodies of the present invention comprising specific embodiments and variants thereof designated as 97A9, 2B2 / 97A9, 54B12 / 163D2, 54B12 / 163E3, 54B12 / 97A9, 97A9 / 127D1, 163D2 / 2B2 or 163E3 / Is at least one sequence optimized amino acid substitution in its framework region and the framework region can be partially or fully humanized, for example. The degree of sequence optimization is at least 80-90% sequence identity to the framework regions with sequences 58, 59, 62, 63, 64, 65, 47, 61, 53, 54, 46, 69, 68, It is desirable to produce a paratrope-like nanobody.

Embodiments of the present invention include methods wherein the first antigen binding domain is selected from polypeptides having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NOS 213, 214, 216 and 219 or one of them, Wherein the antigen binding domain is selected from polypeptides having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NOS: 215, 217 and 218, or one of them.

(0079-0076)

In one embodiment of the invention, the polypeptide comprises a first variable domain in which the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of the single variable domain comprising an immunoglobulin and wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 237 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . The amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 141, 236, 181, 146, 237 or 186. In a further embodiment, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence that differs only in conservative amino acid changes from those set forth in SEQ ID NO: 141, 236, 181, 146, 237 or 186.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 216 or SEQ ID NO: 216, and SEQ ID NO: 217, A second antigen binding domain selected from a polypeptide having at least 80% identity, such as at least 90% identity, e.g., at least 95% identity.

In another further embodiment, the polypeptide comprises SEQ ID NO: 221.

(0079-0086)

In one embodiment of the invention, the polypeptide comprises a first variable domain in which the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of a single variable domain comprising an immunoglobulin, wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 145 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . In a further embodiment, the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 141, 236, 181, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence that differs only in conservative amino acid changes from those set forth in SEQ ID NO: 141, 236, 181, 145, 165 or 185.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 216 or SEQ ID NO: 216, and SEQ ID NO: 218 or SEQ ID NO: 218 A second antigen binding domain selected from a polypeptide having at least 80% identity, such as at least 90% identity, e.g., at least 95% identity.

In yet another further embodiment, the polypeptide comprises SEQ ID NO: 222.

(0079-0061)

In one embodiment of the invention, the polypeptide comprises a first variable domain in which the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of a single variable domain comprising an immunoglobulin, wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 143 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 235 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . In a further embodiment, the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 141, 236, 181, 143, 235 or 183, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence that differs only in conservative amino acid changes from those set forth in SEQ ID NO: 141, 236, 181, 143, 235 or 183.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 216 or SEQ ID NO: 216, A second antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 215, or SEQ ID NO: 215.

(0104-0076)

In one embodiment of the invention, the polypeptide comprises a first variable domain having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 171, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of the single variable domain comprising an immunoglobulin and wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 237 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . In a further embodiment, the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 151,171,191,146, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence that differs only in conservative amino acid changes from those shown in SEQ ID NO: 151, 171, 191, 146, 237 or 186.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 219 or SEQ ID NO: 219, and a first antigen binding domain selected from SEQ ID NO: 217 or SEQ ID NO: 217 A second antigen binding domain selected from a polypeptide having at least 80% identity, such as at least 90% identity, e.g., at least 95% identity.

In yet another further embodiment, the polypeptide comprises SEQ ID NO: 223.

(0104-0086)

In one embodiment of the invention, the polypeptide comprises a first variable domain having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 171, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of a single variable domain comprising an immunoglobulin, wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 145 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . In a further embodiment, the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 151, 171, 191, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence differing only in conservative amino acid changes from those set forth in SEQ ID NO: 151, 171, 191, 145, 165 or 185.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 219 or SEQ ID NO: 219 and a second antigen binding domain selected from SEQ ID NO: 218 or SEQ ID NO: 218 A second antigen binding domain selected from a polypeptide having at least 80% identity, such as at least 90% identity, e.g., at least 95% identity.

In yet another further embodiment, the polypeptide comprises SEQ ID NO: 224.

(0104-0061)

In one embodiment of the invention, the polypeptide comprises a first variable domain having a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 171, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: The first immunoglobulin of a single variable domain comprising an immunoglobulin, wherein the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 143 and the CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 235 and the CDR6 comprises the amino acid sequence set forth in SEQ ID NO: . In a further embodiment, the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80%, such as at least 90% identical to any one of the amino acid sequences set forth in SEQ ID NO: 151, 171, 191, 143, 235 or 183, , E. G. At least 95% amino acid identity.

In a further embodiment, the polypeptide comprises an amino acid sequence that differs only in conservative amino acid changes from those set forth in SEQ ID NO: 151, 171, 191, 143, 235 or 183.

In a further embodiment, the polypeptide comprises a first antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 219 or SEQ ID NO: 219, A second antigen binding domain selected from a polypeptide having at least 80%, such as at least 90%, such as at least 95% identity to SEQ ID NO: 215, or SEQ ID NO: 215.

In the above discussion of preferred embodiments according to the present invention, the disclosure is specifically directed to a bi-parathetic nano-body, but it is also directed to a method wherein the first and second binding domains are selected from the group consisting of conventional 4-chain antibodies, It will be understood that the invention also relates to a double-paratope polypeptide that acts on, or binds to, CXCR2, which is included in chain Fv, Fab, or Fab (2) but has one or more functional or structural characteristics of the preferred embodiments.

It is also contemplated that the first and second antigen binding domains are comprised within immunoglobulin single variable domains such as the V _L domain, the V _H domain, (dAb) and V _HH domains and fragments thereof, and that one or more functional or structural &Lt; / RTI &gt; are clearly disclosed.

In another aspect, the present invention relates to polypeptides that are monovalent to CXCR2 binding and are building blocks for the double paratope polypeptides of the present invention and can be regarded as intermediates in the method of production thereof, particularly immunoglobulin single variable domains such as V _HH Domains or nanobodies. Preferred monovalent immunoglobulin single variable domains include polypeptides having the sequences 25 to 43 and 90 set forth in Table 9 or polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% amino acids in any one of SEQ ID NOS: Is a polypeptide having sequence identity.

Preferred monovalent polypeptides are those designated 137B7 comprising the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with SEQ ID NO: In a preferred embodiment, the framework region of SEQ ID NO: 36 has at least one sequence optimized amino acid substitution. Other preferred monovalent polypeptides are designated 127D1, 2B2, 54B12, 97A9, 163D2, and 163E3, including sequence optimized within the framework region.

For example, 127D1 may comprise the amino acid sequence of SEQ ID NO: 37 where one or more sequence optimized amino acid substitutions are considered in Table 26, and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 216.

2B2 may comprise the amino acid sequence of SEQ ID NO: 43 where one or more sequence optimized amino acid substitutions are considered in Table 20 and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 213 or 214.

54B12 may comprise the amino acid sequence of SEQ ID NO: 90 in which one or more sequence optimized amino acid substitutions are considered in Table 30, and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 219.

97A9 may comprise the amino acid sequence of SEQ ID NO: 39, wherein one or more sequence optimized amino acid substitutions are considered in Table 22, and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 215.

163D2 may comprise the amino acid sequence of SEQ ID NO: 41 where one or more sequence optimized amino acid substitutions are considered in Table 28, and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 218.

163E3 may comprise the amino acid sequence of SEQ ID NO: 42 in which one or more sequence optimized amino acid substitutions are considered in Table 24, and preferably the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 217.

Also provided is a monovalent polypeptide capable of cross-blocking the binding of a polypeptide having any one of the amino acid sequences set forth in SEQ ID NOS: 58, 59, 62, 63, 64, 65, 47 or 61 to CXCR2, in particular an immunoglobulin single variable domain Such as nanobodies, are included in this aspect of the invention.

Any of the preferred monovalent nanobodies discussed above, and in particular 137B7, may be used for the treatment referred to herein, for example, for the treatment of COPD.

The dual paratopoietic polypeptides according to the invention, in particular all the camelised and humanized versions thereof, in particular the preferred dual paratopoietic immunoglobulin single variable domains discussed above, are modulators of CXCR2 and in particular inhibit CXC2 signaling.

Preferably, the CDR sequences and FR sequences in the double paratopoietic polypeptide, particularly the double paratope immunoglobulin single variable domain of the invention,

(K _D ) of 10 ^-5 to 10 ^-12 mol / liter or less, preferably 10 ^-7 to 10 ^-12 mol / liter, more preferably 10 ^-8 to 10 ^-12 mol / (K _A ) of 10 ⁵ to 10 ¹² liters / mole or greater, preferably 10 ⁷ to 10 ¹² liters / mole or greater, and more preferably 10 ⁸ to 10 ¹² liters / mole, to CXCR2 /do or:

10 ² M ^-1 s ^-1 to about 10 ⁷ M ^-1 s ^-1 , preferably 10 ³ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , more preferably 10 ⁴ M ^-1 s Binding to CXCR2 at a k _on rate of ^-1 to 10 ⁷ M ^-1 s ^-1 , such as 10 ⁵ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 ; and / or;

^{_{1 s -1 (t 1/2}} = 0.69 s) to about ^{_{10 -6 s -1 (t 1/}} 2 The number of days muscle ratio providing complex regional (near irreversible)), preferably from 10 ^-2 s ^-1 to to 10 ^-6 s ^-1, to couple to and more preferably 10 ^-3 s ^-1 to 10 ^-6 s ^-1, for example 10 ^-4 s ^-1 to about 10 ^-6 s ^-1 CXCR2 of a k _off rate .

Preferably, the CDR sequences and FR sequences present in the polypeptides of the present invention and in the dual paratopoietic immunoglobulin single variable domains are selected so that they are less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, Lt; RTI ID = 0.0 &gt; CXCR2. &Lt; / RTI &gt;

In particular, as presented in the examples herein, the preferred dual parathetic nanobodies of the present invention are capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM. The preferred dual parathetic nanobodies according to the present invention are also capable of inhibiting the release of agonist (Gro-a) Ca from CXCR2 bearing RBL cells with an IC50 of less than 100 nM. The preferred dual parathetic nanobodies according to the present invention are also capable of inhibiting Gro-a [ ³⁵ S] GTPyS accumulation in the CXCR2-CHO membrane with an IC50 of less than 50 nM. The preferred dual paratopoic nanobodies of the present invention can also inhibit human leukocyte morphological changes at Gro-a exposure to an IC50 of less than 1 nM or to a cynomolgus leukocyte morphology change of less than 2 nM.

According to a most preferred aspect of the present invention, the double-paratope polypeptides of the present invention, such as the double-paratope immunoglobulin single variable domains, such as the nanobodies described herein, may comprise any or all of the sequences 58, 59, 62, 63, 64, 65, 47, or 61, of a polypeptide having the amino acid sequence of SEQ ID NO: 1. Cross-blocking can be measured by any method known to those skilled in the art.

For pharmaceutical uses, the polypeptides of the invention preferably act on a human CXCR2, e. G., A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; For numerical purposes, the polypeptides of the invention preferably act on CXCR2 from the treated species, or are at least cross reactive with CXCR2 from the treated species.

Additionally, the dual paratoptic polypeptides of the invention optionally comprise one or more additional binding sites or domains for binding to other epitopes, antigens, proteins or targets, as well as at least two antigen binding domains for binding to CXCR2, . &Lt; / RTI &gt;

The efficacy of the polypeptides of the present invention, and compositions comprising them, can be assessed by any suitable test known per se suitable for indicating that the polypeptide may be useful in the treatment of COPD or any other disease involving abnormal CXCR2 signaling In vitro assays, cell-based assays, in vivo assays and / or animal models, or any combination thereof. Suitable black and animal models will be apparent to those skilled in the art.

Also, according to the present invention, a polypeptide that acts on human CXCR2 may or may not show cross reactivity with CXCR2 from one or more other species of a warm-blooded animal. Preferably, however, polypeptides of the invention that act on human CXCR2 may be used for toxicity testing in one or more other species of primates such as, but not limited to, monkeys from the genus Macaca (e.g., The Gus Monkey ( Macaca Fasciculus fascicularis ) and / or rhesus monkey ( Macaca mulatta )) and baboons ( Papio ursinus ))). &lt; / RTI &gt; The preferred cross-reactivity is cross-reactivity with CXCR2 from Cynomolgus monkeys. Cross reactivity with animal models of disease (e. G., Mice, rats, mice, pigs or dogs), and in particular with one or more species frequently used in animal models for diseases and disorders associated with CXCR2 may be desirable . In this regard, it will be apparent to those skilled in the art that the cross-reactivity, when present, may have advantages in terms of drug development, since it allows the amino acid sequence and polypeptide acting on human CXCR2 to be tested in the disease model.

More generally, the polypeptides of the present invention that are cross-reactive with CXCR2 from multiple species of mammals will generally be beneficial for use in veterinary applications since they will allow the same polypeptide to be used across multiple species.

Preferably, the double-paratope polypeptide of the invention is not cross-reactive with CXCR1 or CXCR4.

In the double-paratope polypeptides of the present invention, the at least one antigen binding site can act on an interaction site, i.e., a site where CXCR2 interacts with another molecule, e.g., its natural ligand or ligands.

The double paratope polypeptides of the invention, e. G., Immunoglobulin single variable domains, comprise a peptide as shown in SEQ ID NO: 8, 9, 10, 11 or 12, wherein the second antigen binding domain does not bind to the linear peptide of SEQ ID NO: 7 To recognize the epitope present therein. In addition, the first antigen binding domain can comprise an epitope present in or comprising the peptide of SEQ ID NO: 7.

In an embodiment of the invention that cross-reacts with Cynomolgus monkey CXCR2, the first antigen binding domain also includes an epitope that contains or is present within the sequence of SEQ ID NO: 4. In this embodiment, the second antigen binding domain comprises or is capable of recognizing an epitope present in the peptide of SEQ ID NO: 5 or 6.

Also, all naturally occurring or synthetic analogues, variants, mutants, alleles, fragments, and fragments of CXCR2 in general; Or analogs of CXCR2 that contain at least one epitope or epitope essentially identical to the epitope (s) or epitope (s) of the epitope (s) in the CXCR2 (eg, in wild-type CXCR2 of SEQ ID NO: 1) to which the polypeptide of the invention binds. , Variants, alleles, fragments and double-paratovenous polypeptides that bind to fragments are provided within the scope of the present invention. In this case, the polypeptides of the present invention may be modified to have affinity and / or specificity that is the same or different (i. E. Higher or lower) than the affinity and specificity discussed above, to which the polypeptide of the invention binds (wild type) CXCR2. Analogs, variants, mutants, alleles, fragments and fragments thereof.

In addition, as will be apparent to those skilled in the art, the double-paratope conjugated polypeptides bind to CXCR2 with a higher binding force than the corresponding single antigen binding domain polypeptide.

Also, it should be understood that a double-paratovenous polypeptide, particularly a portion, fragment, analog, mutant, variant, allele and / or variant of the dual paratopoietic immunoglobulin single variable domain of the invention, if it always comprises an associated functional domain equivalent to the entire polypeptide It is within the scope of the present invention to use derivatives in the various therapeutic aspects discussed herein. Such moieties, fragments, analogs, mutants, variants, alleles or derivatives may have all of the functional properties discussed above for the double-paratovenous polypeptides of the present invention.

In yet another aspect, the invention relates to a double-paratope polypeptide optionally further comprising one or more other groups, moieties, moieties or binding units, optionally a double-paratope immunoglobulin single variable domain. Such additional groups, moieties, moieties, binding units or amino acid sequences may or may not provide additional functionality to the polypeptides of the invention and may or may not alter their properties.

For example, the additional group, moiety, moiety, or binding unit may be one or more additional amino acid sequences such that the present invention is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, moieties, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, moieties, moieties or binding units are selected from the group consisting of domain antibodies, amino acid sequences suitable for use as domain antibodies, single domain antibodies, amino acid sequences suitable for use as single domain antibodies, " an amino acid sequence suitable for use as a dAb, or a nano-body.

Alternatively, the group, moiety, moiety or linking unit may be a chemical group, moiety, moiety that may, for example, be either biologically and / or pharmacologically active or invisible on its own. For example, the group may be linked to one or more polypeptides of the invention to provide "derivatives" of the polypeptides of the invention as further described herein.

In such constructs, one or more of the polypeptides of the present invention and one or more groups, moieties, moieties or binding units may be linked directly to one another and / or via one or more suitable linkers or spacers. For example, when one or more groups, residues, moieties or binding units are amino acid sequences, the linker may be an amino acid sequence such that the resulting construct is a fusion (protein) or fusion (polypeptide).

As will be apparent from the foregoing and the further description hereof, this means that the double-paratope polypeptides of the present invention can be used in a multi-paratope and optionally multi- or multispecific, bimodal / multivalent and dual / Means that it can be used as a "building block" for forming additional polypeptides of the invention by suitably combining it with other groups, moieties, moieties or binding units to form a structure.

Polypeptides of this aspect of the invention generally comprise at least one step of suitably linking one or more polypeptides of the invention via one or more additional groups, moieties, moieties or binding units, optionally one or more suitable linkers &Lt; / RTI &gt;

In one specific aspect of the invention, the double-paratope polypeptides of the present invention are modified to have an increased half-life compared to the corresponding unmodified polypeptide of the present invention. Certain preferred polypeptides will be apparent to those of ordinary skill in the art based on the further disclosure herein, and may be chemically modified (e. G., Pegylated, pasylated or hesated RTI ID = 0.0 &gt; hesylation) &lt; / RTI &gt; comprising the amino acid sequence or polypeptide of the invention; The polypeptides of the invention may comprise at least one additional binding site for binding to a serum protein (e.g., serum albumin); Or the polypeptides of the present invention may comprise at least one amino acid sequence linked to at least one moiety (and in particular at least one amino acid sequence) which increases the half life of the polypeptide of the invention. Examples of polypeptides of the invention that comprise a moiety or amino acid sequence that extends the half-life include those that bind to one or more serum proteins or fragments thereof, such as (human) serum albumin or a suitable fragment thereof, (E. G., An amino acid sequence suitable for use as a domain antibody, a single domain antibody, an amino acid sequence suitable for use as a single domain antibody, a "dAb &quot;, an amino acid sequence suitable for use as a dAb, Such as a serum albumin (e.g., human serum albumin), a serum immunoglobulin, such as IgG, or a nanobody capable of binding to transferrin; an Fc portion (e.g., human Fc) . In addition, (See, for example, but not limited to, WO 91/01743, WO 01/45746, WO 02/076489, and U.S. Provisional Patent Application Serial No. (See also PCT / EP2007 / 063348)) as described in "Peptides capable of binding to serum proteins").

One of the most widely used techniques for increasing the half-life and / or increasing immunogenicity of pharmaceutical proteins is the use of suitable pharmacologically acceptable polymers such as poly (ethylene glycol) (PEG) or derivatives thereof such as methoxypoly mPEG). &lt; / RTI &gt;

Generally, any suitable form of pegylation can be used, such as pegylation used in the art for antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv); See, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); [Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and WO 04/060965. In addition, various reagents for the pegylation of proteins are commercially available, for example from Nektar Therapeutics, USA.

Preferably, site directed pegylation, particularly through cysteine-residues, is used (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For example, for this purpose, PEG may be attached to naturally occurring cysteine residues in the dual parathetic nanobodies of the invention. The double paratope polypeptides of the present invention can all be modified to suitably introduce one or more cysteine residues for attachment of PEG using protein manipulation techniques known to those of skill in the art or can be modified to incorporate one or more cysteine residues May be fused to the N- and / or C-terminus of the double-paratope polypeptide.

Preferably, for the dual paratopoietic immunoglobulin single variable domains and polypeptides of the present invention, the molecular weight of the PEG used is greater than 5000, such as greater than 10,000 and less than 200,000, such as less than 100,000; For example, in the range of 20,000-80,000.

Pegylation may be applied to one or both of the immunoglobulin variable domains and / or to any peptide linker region. Suitable pegylation techniques are described in EP 1639011.

As an alternative to PEG, the half life may be extended by a technique known as HES involving the attachment of a hydroxyethyl starch (HES) derivative to a polypeptide of the invention. The hydroxyethyl starch used is amylopectin derived from a corn starch which is modified by acid hydrolysis to adjust the molecular weight and the glucose residue therein is hydroxyethylated. Further details can be obtained from Pavisic R, et al., Int J Pharm (2010) March 15, 387 (1-2): 110-9.

Generally, a polypeptide of the invention having an increased half-life is preferably at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, such as at least 10-fold or 20-fold greater than the half-life of the corresponding polypeptide of the invention itself Has a longer half-life of more than twice. For example, a polypeptide of the invention having an increased half-life may have an activity of greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24 hours compared to the corresponding polypeptide of the invention itself , 48 or 72 hours. &Lt; / RTI &gt;

In a preferred aspect of the invention, the polypeptide of the present invention has an amino acid sequence that is greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even 24, 48 or 72 hours, &lt; RTI ID = 0.0 &gt;

In another preferred aspect of the invention, the polypeptides of the present invention exhibit a serum half-life of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours in humans. For example, a polypeptide of the invention may be administered to a subject for at least 5 days (e.g., about 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days Day), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days), or more than 14 days (such as about 14 to 19 days) Lt; / RTI &gt;

The present invention further relates to methods of making or producing the polypeptides, nucleic acids, host cells and compositions of the invention described herein.

In general, these methods

a) providing a set, collection or library of polypeptides; And

b) screening for an amino acid sequence capable of binding to and / or having affinity for a set, aggregate or library of said polypeptide to CXCR2; And

c) isolating the amino acid sequence which binds to and / or has affinity for CXCR2

. &Lt; / RTI &gt;

A set, aggregate or library of polypeptides may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naive set, collection or library of immunoglobulin sequences; A synthetic or semi-synthetic set, aggregate or library of immunoglobulin sequences; And / or a set, aggregate or library of immunoglobulin sequences applied to affinity maturation.

Also, in such methods, a set, aggregate or library of polypeptides may be a set, assembly or library of heavy chain variable domains (e. G., V _H or V _HH domains) or light chain variable domains. For example, a set, aggregate, or library of polypeptides can be a set, aggregate, or library of domain antibodies or single domain antibodies, or can be a set, aggregate, or library of amino acid sequences that can function as domain antibodies or single domain antibodies have.

In a preferred aspect of the method, the set, aggregate or library of polypeptides is suitable, for example, as a CXCR2 or a suitable antigenic determinant derived therefrom, such as its antigenic portion, fragment, region, domain, loop or other epitope Aggregate or library of immunoglobulin sequences derived from a mammal, e.

In such a method, a set, collection or library of peptides or polypeptides can be displayed on a phage, phagemid, ribosome or a suitable microorganism (e.g., yeast) to facilitate screening, for example. Methods, techniques and host organisms suitable for the display and screening of amino acid sequences (sets, assemblies or libraries) will be apparent to those skilled in the art based on, for example, the additional disclosure herein. See also Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In yet another aspect, a method for producing a polypeptide for use in the construction of a double-paratovenous polypeptide according to the present invention comprises

a) providing a cell expressing a population or sample of the polypeptide;

b) screening for a cell expressing a polypeptide capable of binding to and / or having affinity for said aggregate or sample of CXCR2; And

c) (i) isolating said polypeptide; Or (ii) isolating a nucleic acid sequence encoding said polypeptide from said cell, and then expressing said polypeptide

.

For example, when the required polypeptide is an immunoglobulin sequence, the collection or sample of cells may be, for example, a collection or sample of B-cells. Also, in this method, a sample of cells can be obtained from a mammal, for example, a mammal, preferably a human, which has been suitably immunized with CXCR2 or a suitable antigenic determinant based thereon or therefrom, such as an antigenic portion, fragment, region, domain, loop or other epitope derived therefrom , &Lt; / RTI &gt; for example, llama. In one particular aspect, the antigenic determinant may be an extracellular portion, region, domain, loop or other extracellular epitope (s).

In the manufacture of the preferred dual paratope nano-bodies of the invention identified herein, the llama is a mammalian cell that expresses human CXCR2, a mammalian cell that expresses Cynomolgus CXCR2, a DNA that encodes human full-length CXCR2, 17 human DNA encoding CXCR2, DNA encoding Cynomolgus CXCR2, and the peptides shown in Table 5.

The screening method as described above can be performed in any suitable manner as will be apparent to those skilled in the art. See, for example, EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. Screening of step b) is preferably carried out using flow cytometry techniques such as FACS. For this, for example, Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).

In another aspect, a method for producing a polypeptide that acts on CXCR2 for use in the construction of a polypeptide according to the invention comprises at least

a) providing a set, collection or library of nucleic acid sequences encoding the polypeptide;

b) screening for a nucleic acid sequence encoding an amino acid sequence capable of binding to and / or having affinity for a set, aggregate or library of said nucleic acid sequence to CXCR2; And

c) isolating said nucleic acid sequence and then expressing said polypeptide

. &Lt; / RTI &gt;

In such a method, a set, aggregate or library of nucleic acid sequences encoding a polypeptide may be obtained, for example, as a set, collection or library of nucleic acid sequences encoding a set of nests, aggregates or libraries of immunoglobulin sequences; A set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, aggregate or library of immunoglobulin sequences; Aggregation or library of immunoglobulin sequences that are applied to the immune system and / or affinity maturation.

Also, in such methods, a set, aggregate, or library of nucleic acid sequences may encode a set, assembly or library of heavy chain variable domains (e. G. V _H or V _HH domains) or light chain variable domains. For example, a set, aggregate, or library of nucleic acid sequences can encode a set, aggregate, or library of domain antibodies or single domain antibodies, or a set, aggregate, or library of amino acid sequences that can function as domain antibodies or single domain antibodies have.

In a preferred aspect of the method, the set, aggregate, or library of nucleic acid sequences is selected from, for example, CXCR2 or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic portion, fragment, region, domain, loop or other epitope Aggregate or library of nucleic acid sequences derived from a suitably immunized mammal. In one particular aspect, the antigenic determinant may be an extracellular portion, region, domain, loop or other extracellular epitope (s).

In the production of the polypeptides of the invention, the llama was immunized with an antigen as described above.

In such a method, a set, aggregate or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome, or a suitable microorganism (e.g., yeast) to facilitate screening, for example. Methods, techniques, and host organisms suitable for the display and screening of nucleotide sequences (sets, assemblies or libraries of) encoding amino acid sequences will be apparent to those skilled in the art based on, for example, the additional disclosure herein . See also Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, a method of producing a polypeptide that acts on CXCR2, which may be used in a double-paratoptic polypeptide according to the present invention,

a) providing a set, collection or library of nucleic acid sequences encoding the polypeptide;

b) a set, aggregate or library of said nucleic acid sequences can be coupled to and / or have affinity for CXCR2, cross-blocked or hybridized to a double-paratope nano-body of the invention, such as SEQ ID NOS: 58, 59, 62 , 63, 64, 65, 47, or 61, for a nucleic acid sequence encoding an amino acid sequence that cross-blocks the amino acid sequence; And

c) isolating said nucleic acid sequence and then expressing said polypeptide

. &Lt; / RTI &gt;

The invention also relates to a method of determining the nucleotide sequence or amino acid sequence of one of said methods and of said immunoglobulin sequence, by said method or alternatively; The step of expressing or synthesizing said amino acid sequence, and thereby constructing a double-paratovenous polypeptide, by expression in a suitable host cell or host organism, in a manner known per se, or by chemical synthesis, The present invention relates to a double-paratope polypeptide obtained by a method comprising the steps of:

The method may be performed in any suitable manner as will be apparent to those skilled in the art and discussed in more detail below. See, for example, EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. For example, the screening of step b) is preferably carried out using flow cytometry techniques such as FACS. For this, for example, Lieby et al., Blood, Vol. 97, No. 12, 3820]. In particular, reference is made to the so-called "nano clone (TM)" technique described in international patent application WO 06/079372 (Ablingen, et al.

Another technique for obtaining V _HH sequences or nanobody sequences that act on CXCR2 is to appropriately immunize transgenic mammals capable of expressing heavy chain antibodies (i. E., To modulate the immune response and / or to CXCR2 (E.g., a blood sample, a serum sample, or a sample of a B-cell) from the transgenic mammal containing the V _HH sequence or the nucleic acid sequence encoding the nano-body sequence, , And then generating a V _HH sequence that acts against CXCR2, starting from the sample, using any suitable technique known per se (e.g., any method or hybridoma technique described herein) . For example, for this purpose, heavy chain antibody-expressing mice and those described in WO 02/085945, WO 04/049794 and WO 06/008548 and in Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct 10; 103 (41): 15130-5) can be used. For example, the heavy chain antibody expressing mouse can be administered in combination with any suitable (single) variable domain, such as from a natural source (e.g., a human (single), camel (single) or shark (Single) variable domains, and heavy chain antibodies having, for example, synthetic or semi-synthetic (single) variable domains.

Starting from a naturally occurring V _H sequence or preferably a V _HH sequence, other suitable methods and techniques for obtaining a nano-body and / or a nucleic acid encoding it for use in the present invention will be apparent to those skilled in the art, , As described in &lt; RTI ID = 0.0 &gt; WO 08/00279. &Lt; / RTI &gt;

The V _HH domain or nanobody may be characterized by one or more "hallmark residues" within its FR. The hallmark residue is a moiety that is characterized by camelaceous, e. G., FR from a llama source. Thus, the hallmark residue is a preferred target for substitution, preferably for humanization substitution.

According to Kabat numbering, the hallmark residues can be at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 or 108 of the nanobody. Non-limiting examples of (a suitable combination of) said framework sequence and other hallmark residues are set forth in WO 2008/020079, pages 65 to 98, all of which are incorporated herein by reference in their entirety. Other humanized or partially humanized sequences known in the art are also contemplated and are included in the present invention.

As already discussed herein, the nanobodies for use in the present invention have at least one framework region compared to the corresponding framework region of the naturally occurring human V _H domain, At least "one amino acid difference" (as defined herein). More specifically, according to one non-limiting aspect of the present invention, the nanobodies have at least one amino acid substitution compared to the corresponding framework region of the naturally occurring human V _H domain, May have at least "one amino acid difference" (as defined herein) in the hallmark residue (including residues at positions 108, 103 and / or 45). Generally, the nanobodies are formed from a naturally occurring V _H domain at least in one FRM and / or FR4, particularly at least one of the FRM and / or FR4 hallmark residues (again including residues at positions 108, 103 and / or 45) Will have one of the above amino acid differences.

In addition, the humanized nanobodies of the present invention may be as defined herein, but at least one amino acid difference in at least one framework region relative to the corresponding framework region of the naturally occurring V _HH domain Bar). More specifically, a ratio of the present invention, according to a limited aspect, but humanized or may be a different sequence optimized Nanobodies defined herein, at least one as compared to the corresponding framework region of a natural produce V _HH domain At least one amino acid difference "(as defined herein) to the hole-mark residue (including residues at positions 108, 103 and / or 45) In general, a humanized or otherwise sequence-optimized nanobody is capable of binding to at least one of the hallmark residues (again including residues at positions 108, 103 and / or 45) of at least one FR2 and / or FR4, particularly FR2 and / Will have at least one amino acid difference from the naturally occurring V _HH domain.

As will be apparent from the disclosure herein, natural or synthetic analogues, mutants, variants, alleles, homologs and orthologs of the immunoglobulin single variable domains of the invention, as defined herein, Quot;), particularly analogues of the double-parathetic nanobodies of SEQ ID NOS: 58, 59, 62, 63, 64, 65, 47, 61, 53, 54, 46, 69, 68, 67 or 66, .

Generally, in such analogues, one or more amino acid residues may be substituted, deleted and / or added relative to the immunoglobulin single variable domains of the invention as defined herein. The substitution, insertion or deletion may be in one or more framework regions and / or in one or more CDRs. If the substitution, insertion or deletion is in one or more framework regions, they can occur at one or more of the hallmark residues and / or at one or more other positions in the framework residues, but substitution, insertion or deletion Are generally less preferred (unless they are suitable humanized substitutions as described herein).

As a non-limiting example, the substitution may be, for example, conservative substitution (as described herein) and / or the amino acid residue may be replaced with another amino acid residue that is naturally occurring at the same position in another V _HH domain (See WO 2008/020079 for some non-limiting examples of such substitutions), the present invention is generally not limited to this. Thus, for example, it may be desirable to improve the properties of the nanobodies for use in the dual parathetic nanobodies of the present invention, or at least to balance or combine the required or desired properties of the present invention (i. E. Any single substitution, deletion or insertion, or any combination thereof, that does not compromise the double-parathetic nano-body to the extent that it is no longer suitable for its intended use is included within the scope of the present invention. One of ordinary skill in the art will generally be able to determine, based on the teachings herein, of a limited degree of routine experimentation, which may involve, for example, introducing a limited number of possible substitutions and determining its effect on the properties of the resulting nanobodies, After performing, appropriate substitutions, deletions or insertions, or any suitable combination thereof, may be determined and selected.

For example, depending on the host organism used to express the double-paratovenous nanobodies or polypeptides of the present invention, said deletion and / or substitution may comprise one or more sites for post-translational modification (e.g., one or more glycosylation sites) Removed, which can be accomplished by those skilled in the art. Alternatively, substitutions or insertions may be made to introduce one or more sites for attachment of a functional group (as described herein), for example, to allow for site-specific pegylation (again as described herein) Can be designed.

In general, to facilitate substitution, insertion or deletion in an amino acid sequence to support certain characteristics or structural characteristics that are not present in the native sequence, including "humanized" substitution, is referred to as "sequence optimization. In this regard, reference may be made to the definition section of this section of item y).

Analogs are preferably selected so that they have affinity for the double-paratovenic nanobodies of the present invention (K _D -values (actual or apparent), K _A -values (actual or apparent) as defined herein ), k _on -rate and / or k _off -rate, or otherwise suitably measured and / or expressed as an IC50 value).

In addition, the analogs are preferably such that they retain the advantageous properties of the double-parathetic nanobodies as described herein.

According to one preferred aspect, the analogue is selected from the group consisting of one of the double-paratovenous nanobodies of SEQ ID NOS: 58, 59, 62, 63, 64, 65, 47, 61, 53, 54, 46, 69, 68, At least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% sequence identity; Preferably at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only one amino acid difference (as defined herein).

In addition, the framework sequences and CDRs of the analogs are preferably such that they conform to the preferred aspects defined herein. More generally, as described herein, an analogue comprises (a) at position 108; And / or (b) an amino acid or cysteine residue charged at position 45 and E at position 44, and more preferably E at position 44 and R at position 45; And / or (c) have P, R or S at position 103.

One preferred class of analogs of the double-paratovirus V _HH domain or analog of the nanobodies of the invention is humanized (i.e., compared to the naturally occurring nano-body sequence). As mentioned, the humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V _HH with an amino acid residue occurring at the same position in a human V _H domain, such as a human V _H 3 domain. Examples of possible humanized substitutions are specifically disclosed in Tables 20, 22, 24, 26, 28, and 30 of this disclosure, but are not limited to, by comparison of the sequence of the nanobodies with the naturally occurring human V _H domain sequences and in WO 2008/020079 Other combinations of humanized substitutions will be apparent to those skilled in the art.

In general, as a result of humanization, the immunoglobulin single variable domains of the present invention, particularly the nanobodies, can be in a more "human-like" form while still retaining the advantageous properties of the nanobodies of the invention described herein. As a result, the humanized nanobody may have some advantages, such as reduced immunogenicity, as compared to the corresponding naturally occurring V _HH domain. Again, after one on the basis of the disclosure of the present application and optionally to perform routine experimentation in a limited degree, one of ordinary skill in the art the other hand, the advantageous properties and the other provided by the humanizing substitutions required between the natural generation V _HH advantageous properties of the domain Or a suitable combination of humanized or humanized substitutions to optimize or achieve a suitable balance.

The nanobodies for inclusion in the dual parathetic nanobodies of the present invention may be used in any framework residue (s), for example in one or more hallmark residues (as defined herein) or in one or more other framework residues Non-hole-mark residues) or any suitable combination thereof. A preferred humanized substitution for the "P, R, S-103 group" or "KERE family " is the substitution of Q108 with L108. In addition, the nanobodies of the "GLEW class " can be humanized by substitution of Q108 with L108 if at least one other hallmark residue retains camel analogues (as defined herein). For example, as noted above, one particularly preferred class of humanized nanobodies comprises GLEW or GLEW-like sequences at positions 44-47; P, R or S (especially R) at position 103, and L at position 108. [

Humanized and other analogues, and nucleic acid sequences encoding the same, may be provided in any manner known per se, for example using one or more of the techniques mentioned on pages 103 and 104 of WO 08/020079.

As mentioned herein, the immunoglobulin single variable domains (including analogues thereof) of the present invention may be used in combination with the human V _H sequence (including its analogs) to provide the nano-body sequence of the invention and / or to impart advantageous properties of the nano- (I. E., From the amino acid sequence or the corresponding nucleotide sequence), starting from a human V _H 3 sequence, such as DP-47, DP-51 or DP- It will be apparent to those skilled in the art that one or more amino acid residues within the amino acid sequence of the _H domain can be designed and / or manufactured by amino acid residues occurring at corresponding positions of the V _HH domain). Again, this can be done using the various methods and techniques mentioned in the previous paragraph, generally using the amino acid sequence and / or the nucleotide sequence for the human V _H domain as starting points.

Some preferred, but non-limiting, camelizing substitutions can be derived from WO 2008/020079. Also, although camelizing substitutions at one or more of the hallmark residues generally have a greater impact on the desired properties than substitutions at one or more other amino acid positions, both and any suitable combination thereof are within the scope of the present invention Will be included. For example, after introducing one or more camelizing substitutions that already impart at least some of the desired properties, it is possible to introduce additional camelizing substitutions that further improve the properties and / or impart additional advantageous properties Do. Again, if one of ordinary skill in the art are generally based on the disclosure of the present application, and for example, introducing a possible camel Chemistry substitution of a limited number are obtained or improved immunoglobulin advantageous properties of a single variable domain (i.e., compared to the original V _H domain ), It may be possible to determine and select suitable combinations of suitable camelised or camelized substitutions. In general, however, the camelizing substitution is preferably such that the resulting amino acid sequence has at least (a) And / or (b) an amino acid or cysteine residue charged at position 45 and E at position 44, and more preferably E at position 44 and R at position 45; And / or (c) at position 103, P, R or S; And optionally one or more additional camelizing substitutions. More preferably, the camelized substitutions are those in which they are selected from the group consisting of an immunoglobulin single variable domain for use in the invention and / or an analogue thereof (as defined herein), such as a humanized analogue and / To produce an analogue such as a bar.

In addition, immunoglobulin single variable domains, such as nanobodies, can be derived from the V _H domain by introduction of substitutions that are rare in nature, but are nonetheless structurally compatible with V _H domain folds. For example, these substitutions may include one or more of the following: Gly at position 35, Ser, Val or Thr at position 37, Ser, Thr, Arg, Lys, His, Asp at position 39 or Leu, Val, Ala, Thr, or GIu, S or R at position 50 (Barthelemy et al., J Biol Chem. 2008 Feb 8; : 3639-54], [Epub 2007 Nov 28]).

The present invention also includes derivatives of the double paratope polypeptides of the present invention. Such derivatives generally include modifications of the double paratope polypeptides of the present invention and / or one or more amino acid residues that form the double paratope polypeptides of the present invention, especially chemical and / or biological (e.g., by enzymatic) modifications Lt; / RTI &gt;

Examples of such variants, and examples of amino acid residues in the polypeptide sequence that can be modified in this way (i.e. on the protein backbone, but preferably on the side chain), methods and techniques that can be used to introduce such modifications, The potential uses and advantages of the invention will be apparent to those skilled in the art.

For example, the modifications may include one or more functional groups, residues or moieties of the present invention, into or onto the double-paratope polypeptides of the present invention, particularly one or more functional groups, moieties or moieties that impart one or more desired properties or functionality (E. G., By covalent linkage or in other &lt; / RTI &gt; suitable manner) to the double-paratope polypeptides of the present invention. Examples of such functional groups will be apparent to those skilled in the art.

For example, the modifications may increase the half-life, solubility and / or absorption of the polypeptides of the present invention, reduce the immunogenicity and / or toxicity of the polypeptides of the present invention, and reduce any undesirable side effects of the polypeptides of the invention Remove, weaken and / or impart other beneficial properties and / or reduce unwanted characteristics of the dual parathetic nanobodies and / or polypeptides of the present invention; Or introduction of one or more functional groups to provide any combination of the above functions (e.g., by covalent bonding or in any other suitable manner). Examples of such functional groups and techniques for introducing them will be apparent to those of skill in the art and will be understood by those of ordinary skill in the art to be able to modify all functional groups and pharmaceutical proteins generally known in the art and in particular to use antibodies or antibody fragments (including ScFv and single domain antibodies) For example, see Remington ' s Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). The functional group may be linked directly to (e. G., Shared) the double-paratope polypeptide of the invention, e. G., Via a suitable linker or spacer, as would be apparent to one of ordinary skill in the art.

Another, less preferred variant is the use of N-linked or O-linked glycosides of the invention as part of a simultaneous translation and / or post-translational modification, generally in accordance with the host cell used for expressing the double-paratovenous nanobodies or polypeptides of the invention Includes miscalculation.

Another variation may include the introduction of one or more detectable labels or other signal-generators or moieties depending on the intended use of the labeled polypeptide or nanobody. Suitable labels and techniques for attachment, use and detection thereof will be apparent to those skilled in the art and include, for example, fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, radioisotopes, metals, metal chelates, metal cations, Enzymes such as those mentioned on page 109 of WO 08/020079, but are not limited thereto. Other suitable labels will be apparent to those skilled in the art and include moieties that can be detected using, for example, NMR or ESR spectroscopy.

The labeled double-paratope nano-bodies and polypeptides of the present invention can be tested in vitro, in vivo or in situ (depending on the choice of specific label, for example, immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays" and the like) and for in vivo diagnostic and imaging purposes.

As will be apparent to those skilled in the art, another variation may involve introduction of a chelating group, for example, to chelate one of the above-mentioned metal or metal cations. Suitable chelating agents include, but are not limited to, for example, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Another variation may involve the introduction of a functional group that is part of a specific binding pair, e.g., a biotin- (strept) avidin binding pair. The functional groups may be used to link the double-paratope polypeptides or nanobodies of the present invention to another protein, polypeptide or compound that is attached to the other half of the binding pair, i. E. Through the formation of a binding pair. For example, the double-paratovenous nanobodies of the invention may be conjugated to biotin and conjugated to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, the conjugated double-paratovenous nanobodies can be used as a reporter, for example, in a diagnostic system in which a detectable signal-generating agent is conjugated to avidin or streptavidin. The binding pair may be used, for example, to bind the double parathetic nanobodies of the invention to a carrier comprising a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). The binding pair can also be used to link the therapeutic active to the nanobodies of the present invention.

For some uses, in particular for the purpose of killing (for example in the treatment of cancer) cells expressing a CXCR2 target, to which the dual paratopoietic polypeptide or immunoglobulin single variable domain of the invention acts, And / or for purposes of reducing or delaying proliferation, the dual paratopoietic polypeptides of the invention may also be linked to toxins or toxic moieties or moieties. For example, examples of toxic moieties, compounds or moieties that may be linked to the double paratope polypeptides of the present invention to provide cytotoxic compounds will be apparent to those skilled in the art and are described, for example, in the cited prior art and / Can be seen in the further description of the present application. One example is the so-called ADEPT (TM) technology described in WO 03/055527.

Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. The modifications may also be introduced for research purposes (e.g., to study functional-activity relationships). See, for example, Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are selected so that they are compatible with the affinity defined herein for the dual parathetic nanobodies of the present invention (K _D -values (actual or apparent), K _A -values To be expressed and / or expressed suitably as an apparent value), k _on -rate and / or k _off -rate, or alternatively as an IC ₅₀ value.

As mentioned above, the invention also relates to a protein or polypeptide consisting essentially of or comprising at least one of the double-paratope polypeptides of the present invention. "Essentially occurring" means that the amino acid sequence of the polypeptide of the invention is exactly the same as, or a limited number of amino acid residues of the double-paratope polypeptide of the invention, such as 1-20 amino acid residues, 10 amino acid residues, preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues at the amino terminal end, at the carboxy terminal end, or the amino acid sequence of the double-paratope polypeptide Quot; means to correspond to the amino acid sequence of the polypeptide of the present invention added to both the amino terminal end and the carboxy terminal end of the polypeptide.

The amino acid residue may alter, alter, or otherwise not affect (biological) properties of the polypeptide, and may or may not add additional functionality thereto. For example, the amino acid residue may be:

E.g., an N-terminal Met residue as a result of expression in, for example, a heterologous host cell or host organism.

Upon signaling, a signal or leader sequence may be formed from the host cell that directs secretion of the double-paratope polypeptide. Suitable secretion leader peptides will be apparent to those skilled in the art and may be further described herein. Typically, the leader sequence will be linked to the N-terminus of the double-paratope polypeptide;

- allowing the double-paratope polypeptide to induce and / or permit the permeation or introduction of a particular organs, tissues, cells, or portions or compartments of cells, and / or allowing the double-paratope polypeptide to interact with biological barriers such as cell membranes, E. G., A layer of epithelial cells, a tumor comprising solid tumors, or a sequence or signal that allows it to penetrate or cross the blood-brain-barrier. Examples of such amino acid sequences will be apparent to those skilled in the art and include those mentioned in paragraph c) of page 112 of WO 08/020079.

For example, an affinity technique that works on the sequence or residue may be used to form a "tag ", such as an amino acid sequence or residue, that allows or facilitates the purification of a bi-parathetic nano-body. Subsequently, the sequence or residue can be removed (e. G., By chemical or enzymatic cleavage) to provide a double-paratope polypeptide sequence (for this purpose, the tag is cleaved through a cleavable linker sequence to form a double- Or may contain motifs that are optionally linked to or are capable of cleaving sex polypeptide sequences). A preferred, but non-limiting example of a portion of such a residue is a plurality of histidine residues, glutathione residues and a myc-tag (see for example SEQ ID NO: 31 of WO 06/12282).

- may be one or more amino acid residues capable of functioning as attachment sites for functionalized and / or functional groups. Suitable amino acid residues and functional groups will be apparent to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups referred to herein for the double paratope polypeptides or derivatives of the nanobodies of the invention.

According to another aspect, a double-paratope polypeptide of the invention comprises a double-paratope nano-body of the invention and a fusion protein comprising at least one additional peptide or polypeptide, at its amino terminal end, its carboxy At least one additional peptide or polypeptide at its terminal end, or both at its amino terminal end and its carboxy terminal end. The fusants will also be referred to herein as "nanobody fusions ".

Preferably, the additional peptides or polypeptides are intended to impart one or more of the desired properties or functionality to the double-parathetic nanobodies or polypeptides of the invention.

For example, additional peptides or polypeptides may also provide additional binding sites, which may be any desired proteins, polypeptides, antigens, antigenic determinants, or epitopes, including the double-paratope polypeptides of the present invention Including but not limited to the same proteins, polypeptides, antigens, antigenic determinants or epitopes acting on, or different proteins, polypeptides, antigens, antigenic determinants or epitopes acting on.

Examples of such peptides or polypeptides will be apparent to those skilled in the art and include all amino acid sequences commonly used in peptide fusions based on antibodies and fragments thereof (including, but not limited to, ScFv and single domain antibodies) . See, for example, Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, the peptides or polypeptides may be used to increase half-life, solubility, or absorption of the polypeptides of the invention, reduce immunogenicity or toxicity, eliminate or attenuate undesirable side effects, and / Or to impart other beneficial properties and / or to reduce unwanted characteristics. Non-limiting examples of such peptides and portions of polypeptides include serum proteins, such as human serum albumin (see for example WO 00/27435) or hapten molecules (e. G., Haptens recognized by circulating antibodies, e. / 22141).

In particular, it is described in the prior art that linking fragments of immunoglobulins (e.g., the V _H domain) to serum albumin or fragments thereof can be used to increase half-life. WO 00/27435 and WO 01/077137. According to the present invention, a dual paratopoietic polypeptide, preferably a dual paratovenic nano-body of the invention, preferably comprises a sequence complementary to serum albumin (or a suitable fragment thereof) or a polypeptide of the invention as a gene fusion protein Lt; / RTI &gt; through a suitable linker so as to be able to be expressed. According to one particular aspect, the double-paratovenous nanobodies of the present invention can be linked to fragments of serum albumin that comprise at least domain III of serum albumin or a portion thereof. See, for example, WO 07/112940 to Ablingen, et al.

Alternatively, as already discussed herein, additional peptides or polypeptides may be used that act on serum proteins (e. G., Human serum albumin or other serum proteins, e. G., IgG) to provide an increased half- To provide additional binding sites or binding units. Such amino acid sequences may be found, for example, in the nano-bodies described below, and the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAbs described in WO 03/002609 and WO 04/003019 . See also Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, and EP 0 368 684, and WO 08/028977, WO 08/043821, WO 08/043822 (Ablingen, V.), and Ablingen, (Also referred to as " Peptides capable of binding to serum proteins ") (also see PCT / EP2007 / 063348).

The peptides or polypeptides may specifically act on serum albumin (more particularly human serum albumin) and / or IgG (more particularly human IgG). For example, the amino acid sequence may include an amino acid sequence that acts on (human) serum albumin and an amino acid sequence capable of binding to an amino acid residue on (human) serum albumin that is not involved in binding to FcRn of serum albumin WO 06/0122787) and / or an amino acid sequence capable of binding to an amino acid residue on the serum albumin which does not form part of the domain III of the serum albumin (again see, for example, WO 06/0122787); An amino acid sequence that may have or provide an increased half-life (see, e. G., WO 08/028977 to Ablingen, et al.); At least one species of mammal, and in particular at least one species of primate, such as, but not limited to, monkeys from the genus Macaca (e.g., and in particular Cynomolgus monkeys (macaca fascicularis) and / (See again WO 08/028977) which acts on human serum albumin which is cross-reactive with serum albumin from monkeys (macaca mulatta) and baboons (papiouracinus)); Amino acid sequences capable of binding to serum albumin in a pH-independent manner (see, for example, WO 08/043821 (abbreviated as "Amino acid sequences that bind to serum proteins in a (see, for example, WO 08/043822 (entitled "Amino acid sequences that bind to ") and / or conditional binders a desired molecule in a conditional manner ")).

According to another aspect, the one or more additional peptides, polypeptides or protein sequences may comprise one or more portions, fragments or domains of conventional four chain antibodies (particularly human antibodies) and / or heavy chain antibodies. For example, while less preferred, the double-paratovenous nanobodies of the present invention may also optionally comprise a linker sequence (including but not limited to other (single) domain antibodies such as the dAbs described in Ward et al. To a conventional (preferably human) V _H or V _L domain or to a natural or synthetic analogue of a V _H or V _L domain.

The double-paratope polypeptides or nanobodies may also optionally be linked to one or more (preferably human) C _H 1, C _H 2 and / or C _H 3 domains via linker sequences. For example, a double-paratovenous nanobody linked to a suitable C _H 1 domain may be one of the common V _H domains or (F (ab ') 2) fragments similar to, for example, conventional Fab fragments or F ₂ fragments) can both be used in conjunction with a light chain suitable for producing an antibody fragment / structure that has been replaced by the double-paratovenous nanobodies of the present invention. In addition, the two double-paratope polypeptides may be linked to a CH3 domain (optionally through a linker) to provide a construct with increased half-life in vivo.

According to one particular aspect of the polypeptide of the present invention, the one or more double-paratope polypeptides or nanobodies of the invention comprise one or more constant domains (e. G., 2 &lt; RTI ID = Fragments or domains that are capable of imparting to the Fc portion and / or one or more effector functions to the polypeptides of the invention and / or to one or more Fc receptors, (Optionally through a suitable linker or hinge region). For example, for this purpose, and without limitation, one or more additional peptides or polypeptides may be conjugated to an antibody, such as a heavy chain antibody (as described herein), more preferably from a conventional human 4-chain antibody And / or may comprise at least one C _H2 and / or C _H2 domain; (Part of) the Fc region from, for example, an IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgE or another human Ig, such as IgA, IgD or IgM. For example, WO 94/04678 describes a heavy chain antibody comprising a camelid V _HH domain or a humanized derivative thereof (i.e., a nanobody), wherein the camelidae C _H 2 and / or C _The H3 domain is composed of human C _H 2 and C _H 2 to provide an immunoglobulin consisting of two heavy chains, each containing a nano-body and human C _H 2 and C _H 3 domains (but not a C _H 1 domain) 3 domain, which immunoglobulin can function even if it has an effector function provided by the C _H 2 and C _H 3 domains and no light chain is present. Other amino acid sequences that may be suitably linked to the nano-bodies of the present invention to provide effector functions will be apparent to those skilled in the art and may be selected based on the desired effector function (s). See, for example, WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, and Holliger and Hudson, supra. The coupling of polypeptides, e. G., To the Fc portion of the nanobodies of the invention, can also lead to increased half-life compared to the corresponding polypeptides of the present invention. For some uses, the use of Fc portions and / or constant domains (i.e., C _H 2 and / or C _H 3 domains) that do not exhibit any biologically significant effector function, but which confer an increased half-life, It may even be desirable. One or more double-paratope polypeptides with increased half-life in vivo, such as nanobodies and other suitable constructs comprising one or more constant domains, will be apparent to those skilled in the art and may, for example, optionally be linked to the C _H 3 domain And may include two nanobodies connected thereto. In general, any fusion protein or derivative with an increased half-life will preferably have a molecular weight greater than 50 kD, a cut-off value for renal absorption.

In yet another specific but non-limiting aspect, in order to form a polypeptide of the invention, the one or more amino acid sequences of the present invention have a reduced (or essentially non-existent) tendency to self associate to a dimer (Optionally through a suitable linker or hinge region) to a naturally occurring, synthetic or semi-synthetic constant domain (or an analogue, variant, mutant, part or fragment thereof) relative to a naturally occurring constant domain in a 4 chain antibody. Such monomeric (i.e., non-self-associating) Fc chain variants, or fragments thereof, will be apparent to those skilled in the art. For example, Helm et al., J Biol Chem 1996 271 7494 describes monomeric Fc chain variants that can be used in the polypeptide chains of the present invention.

In addition, the monomeric Fc chain variants are preferably designed to allow them to continue to bind complement or related Fc receptor (s) (depending on the Fc portion from which they are derived) and / or to provide a portion of the effector function of the Fc portion Or all (or at a reduced level that continues to be appropriate for the intended use). Alternatively, in the polypeptide chain of the present invention, the monomeric Fc chain can be used to confer increased half-life on the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector function .

Additional peptides or polypeptides may also form signal or leader sequences that direct secretion of the double-paratovenic nanobodies or polypeptides of the invention from the host cell during synthesis (e. G., Expression of the polypeptide of the invention To provide a pre-, pro- or prepro-form of the polypeptide of the invention according to the host cell to which it is to be introduced.

Additional peptides or polypeptides may be used to permit the dual paratope nano-bodies or polypeptides of the present invention to be directed towards and / or permeated into or introduced into a particular organ, tissue, cell, or portion or compartment of a cell, and / It is contemplated that the double-paratovenous nanobodies or polypeptides of the invention may be capable of binding to a biological barrier, such as a cell membrane, a layer of a cell, such as a layer of epithelial cells, a tumor comprising solid tumors, or a sequence or signal that allows it to penetrate or cross the blood- . Suitable examples of such amino acid sequences will be apparent to those skilled in the art and include, but are not limited to, those mentioned, for example, on page 118 of WO 08/020079. For some uses, in particular, the double-paratovenous polypeptides of the present invention may be used to kill (e.g., in the treatment of cancer) cells that express a target acting thereon, or to reduce the growth and / For purposes in which it is intended that the double-paratope polypeptides of the invention are also intended to be linked to (cellular) toxic proteins or polypeptides. For example, examples of such toxic proteins and polypeptides that may be linked to the nanobodies of the present invention to provide cytotoxic polypeptides of the invention will be apparent to those skilled in the art, and are described, for example, in the above cited prior art and / Can be seen in the further description. One example is the so-called ADEPT (TM) technology described in WO 03/055527.

According to one optional but non-limiting aspect, the one or more additional peptides or polypeptides may comprise at least 3, e.g., 4, or more than 5 &lt; RTI ID = 0.0 &gt; nano- At least one additional nanobody to provide a polypeptide of the invention comprising a body.

Finally, it is also within the scope of the present invention that the double-paratope polypeptides of the present invention may comprise two or more nanobodies and one or more additional peptides or polypeptides (as referred to herein).

For polyvalent and multispecific polypeptides and preparations containing two or more V _HH domains, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001); [Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302]; And for example WO 96/34103 and WO 99/23221. Other examples of some of the specific multispecific and / or polyprotein polypeptides of the present invention can be found in the Ablinges, et al. Application, referred to herein.

One preferred example of a multispecific polypeptide of the invention comprises at least one double-paratope nano-body of the invention and at least one nanobody providing an increased half-life. The nanobodies may be used, for example, against serum proteins, especially human serum albumin, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, immunoglobulins such as IgG, IgE or IgM, It may be a nanobody that acts on one of the serum proteins listed in WO 00/003019. Of these, nanobodies capable of binding to serum albumin (especially human serum albumin) or to IgG (particularly human IgG, see for example Nanobody VH-1 described in Muyldermans, supra) are particularly preferred (However, for example, for experiments in mice or primates, a nano-body may be used that is either a mouse serum albumin (MSA) or a nano-body that acts on or is cross-reactive with serum albumin from the primates. , Human serum albumin, or nanobodies that act on human IgG. Nanobodies which provide an increased half-life and can be used in the polypeptides of the present invention are described in further patent applications in WO 04/041865, WO 06/122787 and Ablingen, et al., For example on serum albumin as described above And a nano body.

For example, some preferred nanobodies providing increased half-life for use in the present invention include nanobodies capable of binding to amino acid residues on (human) serum albumin that are not involved in binding to FcRn of serum albumin (e. For example, WO 06/0122787) and / or nanobodies capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see, e.g., WO 06/0122787); Nanobodies capable of providing or providing increased half-life (see, for example, WO 08/028977 to Ablingen, et al., Herein incorporated by reference); At least one species of mammal, and in particular at least one species of primate, such as, but not limited to, monkeys from the genus Macaca (e.g., and in particular Cynomolgus monkeys (macaca fascicularis) and / Nanobodies acting on human serum albumin (see WO 08/028977 to Ablingen, et al.) Which are cross-reactive with serum albumin from monkeys (macaca mulatta) and baboons (papiouracinus) ; (see, e. g., WO 2008/043821 to Ablingans, et al., cited herein) and / or nanobodies (e. g., Ablins See WO &lt; RTI ID = 0.0 &gt; 08/043822 &lt; / RTI &gt;

Some particularly preferred nanobodies that provide an increased half-life and can be used in the polypeptides of the present invention include the nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables II and III), of which ALB -8 (SEQ ID NO: 62 of WO 06/122787) is particularly preferred.

According to a particular aspect of the invention, the polypeptide of the invention contains, in addition to two or more nanobodies, at least one nanobody which acts on human serum albumin.

In addition, additional peptides or polypeptides that may be added, attached or fused to the double paratope polypeptides of the invention include polymers comprised of proline, alanine, and serine (PAS sequence). The PAS sequence consists of 200-600 residues, which can lead to a dramatic increase in hydrodynamic volume and prolong plasma half-life. In addition, the serum half-life of the double-paratope polypeptides of the present invention can be determined for polypeptides of 864 amino acids termed XTEN as described in Schellenbrger et al. (2009), Nature Biotechnology 27, No 12, p1186-1190 Can be extended by fusion.

In general, any polypeptide of the invention having an increased half-life comprising one or more double-paratope nano-bodies of the invention, and any of the double-paratovenous nanobodies of the invention having an increased half- Of the present invention preferably has a greater half-life of at least 1.5 times, preferably at least 2 times, such as at least 5 times, such as at least 10 times or more than 20 times the half-life of the corresponding nanobodies of the invention itself. For example, the derivative or polypeptide having an increased half-life may have an activity of greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24 hours compared to the corresponding polypeptide of the invention itself , 48 or 72 hours. &Lt; / RTI &gt;

In a preferred but non-limiting aspect of the invention, the derivatives or polypeptides have a serum half-life of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours in humans . &Lt; / RTI &gt; For example, the derivative or polypeptide may be administered for at least 5 days (eg, about 5 to 10 days), preferably at least 9 days (eg, about 9 to 14 days), more preferably at least about 10 days Day), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days), or more than 14 days (such as about 14 to 19 days) Lt; / RTI &gt;

Another preferred but non-limiting example of a multispecific polypeptide of the present invention includes at least one double-paratope nano-body of the invention and a polypeptide of the invention in combination with a particular organ, tissue, cell, Such as a cell membrane, a layer of a cell layer such as a layer of epithelial cells, a tumor comprising a solid tumor, or a blood sample, such as blood, - at least one nanobody that allows the brain-barrier to penetrate or cross. Examples of such nanobodies include nanobodies that are directed toward the desired cell-surface proteins, markers or epitopes (e.g., cell-surface markers associated with tumor cells) of the desired organs, tissues or cells, and nanobodies such as those described in WO 02/057445 and WO 06 / 040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the present invention, the two or more nanobodies and the one or more polypeptides may be directly connected to each other and / or through one or more suitable spacer or linker (e. G., As described in WO 99/23221) Can be connected to each other.

According to one aspect of the invention, the polypeptides of the invention are present in essentially isolated form as defined herein.

The amino acid sequences, double-paratope nano-bodies, polypeptides and nucleic acids of the present invention can be prepared in a manner known per se as will be apparent to those skilled in the art from the further description herein. For example, the double-paratovenous nanobodies and polypeptides of the present invention can be used in the manufacture of antibodies, in particular any of the antibodies known for themselves for the manufacture of antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments) . &Lt; / RTI &gt; Some preferred but non-limiting methods for preparing amino acid sequences, nanobodies, polypeptides, and nucleic acids include those described herein.

As will be apparent to those skilled in the art, one method that is particularly useful for the production of the dual parathetic nanobodies and / or polypeptides of the present invention generally comprises the following steps:

i) a suitable host cell or host organism of nucleic acid (also referred to herein as "the nucleic acid of the present invention") encoding said double-paratope nano-body or polypeptide of the invention (also referred to herein as " Or expression in another suitable expression system, and then optionally

ii) Isolation and / or purification of the thus obtained double-parathetic nanobodies or polypeptides of the present invention.

In particular, the method may comprise the following steps:

i) cultivation and / or maintenance of the host of the invention under conditions which allow the host of the invention to express and / or produce at least one of the double-paratope nano-bodies and / or polypeptides of the invention,

ii) Isolation and / or purification of the thus obtained double-parathetic nanobodies or polypeptides of the present invention.

In yet another aspect, the invention is directed to a nucleic acid molecule encoding a polypeptide of the invention (or a suitable fragment thereof). The nucleic acid will also be referred to herein as "the nucleic acid of the invention" and may exist, for example, in the form of a gene construct described further herein.

In a preferred embodiment, the present invention provides a nucleic acid molecule encoding an amino acid sequence selected from the group of amino acid sequences set forth in SEQ ID NOS: 25 to 43, 90 and SEQ ID NOS: 213 to 219 for each particular nanobody in Tables 9 and 32 . Alternatively, the nucleic acid molecule according to the invention comprises nucleic acid molecules encoding the polyvalent and double-parathetic nanobody constructs of SEQ ID NOS: 44-69. In addition, the nucleic acid molecule according to the present invention comprises a molecule having a nucleic acid sequence according to SEQ ID NOS: 192 to 211 relating to the nanobodies identified in Table 18.

The nucleic acid of the present invention may be in the form of single or double stranded DNA or RNA, preferably in the form of double stranded DNA. For example, the nucleotide sequence of the present invention can be a genomic DNA, a cDNA, or a synthetic DNA (e. G., A DNA having a codon usage frequency specifically adjusted for expression in an intended host cell or host organism).

According to one aspect of the present invention, the nucleic acid of the present invention is present in essentially isolated form as defined herein.

The nucleic acid of the present invention may also be present in the form of a vector which may be present again in its essentially isolated form, for example in the form of a plasmid, cosmid or YAC, and / or be present and / or part thereof.

The nucleic acid of the present invention may be prepared or obtained in a manner known per se based on information on amino acids for the polypeptides of the present invention set forth herein and may be isolated from natural sources suitable or suitable. To provide an analog, the nucleotide sequence encoding the naturally occurring V _HH domain may be applied, for example, to site directed mutagenesis to provide a nucleic acid of the invention encoding the analog. In addition, as will be apparent to those skilled in the art, in order to prepare the nucleic acid of the present invention, it is also possible to employ several nucleotide sequences, such as at least one nucleotide sequence encoding the polypeptide of the present invention and a nucleic acid encoding, for example, Can be connected together.

Techniques for generating nucleic acids of the present invention will be apparent to those skilled in the art and include, for example, automated DNA synthesis; Site directed mutagenesis; A combination of two or more naturally occurring and / or synthetic sequences (or a portion of two or more thereof), introduction of a mutation that induces expression of the truncated expression product; The introduction of one or more restriction sites (e.g., for the generation of cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes), and / or for the production of CXCR2 as a template, , And introducing mutations by PCR reactions using one or more "mismatch" primers. These and other techniques will be apparent to those skilled in the art and refer back to standard guides such as the above-mentioned references [Sambrook et al., And Ausubel et al., And the examples below.

The nucleic acid of the present invention may also be present in the form of a gene construct and / or be present within and / or part of it. The gene construct generally comprises one or more elements of the known genetic construct, such as one or more suitable regulatory elements such as suitable promoter (s), enhancer (s), terminator (s) And at least one nucleic acid of the invention optionally linked to an additional element of the gene construct mentioned. The gene construct comprising at least one nucleic acid of the present invention will also be referred to herein as "the gene construct of the present invention ".

The gene construct of the present invention may be DNA or RNA, preferably double-stranded DNA. For example, the gene constructs of the present invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell, / RTI &gt; and / or &lt; / RTI &gt; For example, the gene construct of the present invention may be in the form of a vector, such as a plasmid, a cosmid, a YAC, a viral vector or a transposon. In particular, the vector may be an expression vector, that is, a vector capable of providing expression in vitro and / or in vivo (e.g., in a suitable host cell, host organism, and / or expression system).

In a preferred but non-limiting aspect, the gene construct of the present invention comprises

i) at least one nucleic acid of the invention; Operably connected thereto

ii) one or more regulatory elements such as a promoter and optionally a suitable terminator; And optionally also

iii) one or more additional elements of the genetic construct known per se

;

As used herein, the terms "operably connected" and "operably linked" have the meanings indicated on pages 131-134 of WO 08/020079; &Quot; Regulatory element ", "promoter "," terminator ", and "additional elements" are described on pages 131-134 of WO 08/020079; Genetic constructs can be further described on pages 131-134 of WO 08/020079.

The nucleic acid of the present invention and / or the gene construct of the present invention can be used to transform a host cell or host organism, i. E., The expression and / or production of the double-paratovenous nanobodies or polypeptides of the present invention. Suitable hosts or host cells will be apparent to those skilled in the art and include, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms, such as those described in WO 08/020079 on pages 134 and 135 Described; And any other host or host cell known per se for the expression and production of antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments), which will be apparent to those skilled in the art. In addition, the general background cited above, and for example WO 94/29457; WO 96/34103; WO 99/42077; [Frenken et al., (1998), supra]; [Riechmann and Muyldermans, (1999), supra]; [van der Linden, (2000), supra]; [Thomassen et al., (2002), supra; [Joosten et al., (2003), supra]; [Joosten et al., (2005), supra; And additional references cited herein.

The dual parathetic nanobodies and polypeptides of the present invention may also be used for prophylactic and / or therapeutic purposes, as further described in, for example, pages 135 and 136 of WO 08/020079 and in further references cited in WO 08/020079 (E.g., as a gene therapy) is introduced and expressed in one or more cells, tissues or organs of a multicellular organism.

For expression of nanobodies in cells, they are also described, for example, in WO 94/02610, WO 95/22618 and US-A-7004940; WO 03/014960; Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag]; And as "intrabody" as described in [Kontermann, Methods 34, (2004), 163-170).

In addition, the double-paratovenous nanobodies and polypeptides of the present invention can be used in, for example, transgenic mammals, in mammals, for example, in mice, cows, (See, for example, US-A-6,741,957, US-A-6,304,489, and US-A-6,849,992 for general techniques for the treatment of plants), plants, or parts of plants including, but not limited to leaves, flowers, fruits, seeds, roots, For example in potatoes, corn, soy or alfalfa) or for example in the case of Bombix mori ) can be produced in the pupa.

In addition, the double-paratovenous nanobodies and polypeptides of the present invention may also be expressed and / or produced in a cell-free expression system, and suitable examples of such systems will be apparent to those skilled in the art. Some preferred, but non-limiting examples are malt systems; Rabbit reticulocyte lysate; Or this. Lt; RTI ID = 0.0 &gt; E. coli &lt; / RTI &gt; Zubay system.

As mentioned above, one of the advantages of using double-paratovenous polypeptides and nanobodies is that the polypeptides based thereon can be produced through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells , Regulatory elements, etc. will be apparent to those skilled in the art, for example, from the above-referenced references. It should be noted, however, that the invention is not limited in its broadest sense to expression in bacterial systems.

Preferably, in the present invention, an expression system, such as a bacterial expression system, is used which provides the polypeptide of the present invention in a form suitable for pharmaceutical use, such expression system will again be apparent to those skilled in the art. Also, as will be apparent to those skilled in the art, polypeptides of the present invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For industrial scale production, preferred heterologous hosts for (industrial) production of double-paratovenous nanobodies or nanobody-containing protein therapeutics are those that are used for large scale expression / production / expression, especially for large scale pharmaceutical (ie GMP grade) / Suitable for expression. Coli, Pichia pastoris ), S. Lt; RTI ID = 0.0 &gt; S. cerevisiae. &Lt; / RTI &gt; Suitable examples of such strains will be apparent to those skilled in the art. The strain and production / expression system are also available from companies such as Biovitrum, Uppsala, Sweden.

Alternatively, mammalian cell lines, particularly Chinese hamster ovary (CHO) cells, may be used for large-scale expression / production / expression, particularly for large-scale pharmaceutical expression / production / expression. Again, the expression / production system is also available from some of the companies mentioned above.

The selection of a particular expression system will in part be determined by the specific post-translational modification, more specifically the need for glycosylation. Production of the required nano-body-containing recombinant protein, for which glycosylation is required or required, will require the use of a mammalian expression host having the ability to glycosylate the recombinant protein. In this regard, it will be apparent to those skilled in the art that the resulting glycosylation pattern (i. E., The type, number and location of attached moieties) will be determined by the cell or cell line used for expression. Preferably, a human cell or cell line is used (i. E., To produce a protein having an essentially human glycosylation pattern) or glycosylation that mimics human glycosylation, essentially and / or functionally equivalent to at least human glycosylation Another mammalian cell line capable of providing a pattern is used. Generally, prokaryotic hosts, e. Cola does not have the ability to glycosylate proteins, and low-grade prokaryotes, such as yeast, generally produce a glycosylation pattern that is different from human glycosylation. Nevertheless, it should be understood that all such host cells and expression systems can be used in the present invention in accordance with the required double-paratovenous nanobodies or polypeptides to be obtained.

Thus, according to one aspect of the present invention, the double-parathetic nanobodies or polypeptides of the invention are glycosylated. According to yet another non-limiting aspect of the invention, the amino acid sequence, nanobody or polypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting aspect of the present invention, the double-paratovenous nanobodies or polypeptides of the invention are produced in bacterial cells, particularly bacterial cells suitable for large scale pharmaceutical production, such as cells of the above-mentioned strains.

According to another preferred but non-limiting aspect of the present invention, the double-paratovenous nanobodies or polypeptides of the present invention are produced in yeast cells, particularly yeast cells suitable for large scale pharmaceutical production, such as cells of the above-mentioned species.

According to another preferred but non-limiting aspect of the present invention, the double-paratovenous nanobodies or polypeptides of the present invention may be used in mammalian cells, particularly human cells or cells of human cell lines, more particularly human cells suitable for large- A cell of a human cell line, such as the cell line referred to hereinabove.

When expression in a host cell is used to produce the double-paratovenous nanobodies and polypeptides of the present invention, as further described in pages 138 and 139 of WO 08/020079, they can be expressed in cells (e. G. , Peripheral cytoplasm or inclusion bodies) and then isolated from the host cell and optionally further purified; Or produced extracellularly (e.g., in a medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified. Thus, according to one non-limiting aspect of the present invention, the double-paratovenous nanobodies or polypeptides of the present invention are produced in a cell and isolated from an inclusion body in a host cell, in particular a bacterial cell or a bacterial cell, Body or polypeptide. According to another non-limiting aspect of the present invention, the double-paratovenous nanobody or polypeptide of the invention is a nanobody or polypeptide isolated from a medium produced extracellularly and the host cell is cultured.

Some preferred, but non-limiting, promoters for use with these host cells include those mentioned on pages 139 and 140 of WO 08/020079.

Preferred, but non-limiting, partial secretion sequences for use with these host cells include those mentioned on page 140 of WO 08/020079.

Suitable techniques for transforming a host or host cell of the invention will be apparent to those skilled in the art and may be determined depending on the intended host cell / host organism and the genetic construct used. Again refer to the above mentioned guide and patent application.

Following transformation, the step of detecting and selecting a host cell or host organism that has been successfully transformed with the nucleotide sequence / gene construct of the present invention can be performed. This may be, for example, a selection step based on a selectable marker present in the gene construct of the invention or a step involving the detection of a polypeptide of the invention using, for example, a particular antibody.

Transformed host cells (which may exist in the form of stable cell lines) or host organisms (which may exist in the form of stable mutant strains or strains) form a further aspect of the present invention.

Preferably, these host cells or host organisms are selected so that they are capable of expressing the double-paratovenous nanobodies or polypeptides of the invention (e. G. Under suitable conditions) (and in the case of host organisms: at least one cell, Or (at least) in the organ. The present invention also encompasses additional generations, progeny and / or offspring of the host cell or host organism of the present invention, which may be obtained, for example, by cell division or by oily or asexual propagation.

In order to produce or obtain an expression product of the amino acid sequence of the present invention, the transformed host cell or transformed host organism is generally transformed into a host cell under conditions that allow expression / production of the (desired) double-paratope nano-body or polypeptide of the present invention Maintained, and / or cultured under conditions such as that described above. Suitable conditions will be apparent to those skilled in the art and will be determined by the host cell / host organism commonly used and the regulatory elements controlling the expression of the (related) nucleotide sequences of the invention. Again, reference is made to the guidance and patent applications mentioned in the paragraph on the gene construct of the present invention.

In general, suitable conditions include, but are not limited to, the use of a suitable medium, the presence of a suitable source of food and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g., the nucleotide sequence of the invention, Lt; RTI ID = 0.0 &gt; (e. &Lt; / RTI &gt; All of which may be selected by one of ordinary skill in the art. Again, under the above conditions, the polypeptides of the present invention may be expressed in a constitutive manner, transiently, or only if suitably derived.

It will also be appreciated by those skilled in the art that the double-paratovenous nanobodies or polypeptides of the present invention can be (first) produced in an immature form (as mentioned above) and subsequently applied to a post-translational modification depending on the host cell / host organism used It will be obvious. In addition, the dual parathetic nanobodies or polypeptides of the present invention may be glycosylated according to the host cell / host organism being used again.

The double-paratovenous nanobodies or polypeptides of the present invention may be used as such by known protein isolation and / or purification techniques, such as (preliminary) chromatography and / or electrophoresis techniques, rate differential precipitation techniques, affinity techniques (E. G., Using an amino acid sequence of the invention, a nano-body or a specific cleavable amino acid sequence fused to a polypeptide) and / or preimmunologic techniques (e. G. Using an antibody to the amino acid sequence being isolated) Or the host cell or the host organism can be isolated from the culture medium.

Generally, for pharmaceutical use, the polypeptides of the present invention comprise at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharmaceutical active polypeptide and / Or a pharmaceutical composition or a composition comprising the compound. As a non-limiting example, the formulations can be formulated for oral, parenteral (e.g., by intravenous, intramuscular or subcutaneous or intravenous infusion), topical, by inhalation (e.g., For example, via an inhaler (MDI) or dry powder inhaler (DPI) or through a nasal route), skin patches, implants, suppositories, sublingual routes, and the like. Such suitable dosage forms (which may be solid, semi-solid or liquid depending on the mode of administration) and methods and carriers for use in the manufacture thereof will be apparent to those skilled in the art and are further described herein.

Thus, in a further aspect, the invention provides a kit comprising at least one double-paratope polypeptide of the invention, preferably at least one double-paratope immunoglobulin single variable domain, more preferably at least one double- A parathetic nano-body, and at least one suitable carrier, diluent or excipient (i. E. For suitable pharmaceutical use), and optionally one or more further active substances.

In general, the double paratope polypeptides of the present invention can be formulated and administered in any suitable manner known per se, including the general background art mentioned above (and in particular WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) and a standard guide, for example, the literature ^{[Remington's Pharmaceutical Sciences, 18 th} Ed., Mack Publishing Company, USA (1990)], [Remington, the Science and Practice of Pharmacy , 21th Edition, Lippincott Williams and Wilkins (2005); Or Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

For example, the double-paratope polypeptides of the invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv and diabodies) and other pharmaceutical active proteins. Such formulations and methods of manufacture thereof will be apparent to those of ordinary skill in the art and will be apparent to those skilled in the art, for example, by parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intrathecal, intraarterial, Or &lt; / RTI &gt; the skin).

Formulations for parenteral administration may be, for example, sterile solutions, suspensions, dispersions or emulsions suitable for injection or injection. Suitable carriers or diluents for the formulations include, but are not limited to those mentioned, for example, on page 143 of WO 08/020079. In general, aqueous solutions or suspensions will be preferred.

The dual paratopoietic polypeptides of the invention comprising a double paratope immunoglobulin single variable domain and a nanobody can also be administered using a gene therapy delivery method. See, for example, U.S. Patent 5,399,346, which is incorporated herein by reference in its entirety. By using the gene therapy transfer method, primary cells transfected with the gene encoding the double-paratope or polypeptide of the present invention can be further transformed with a tissue-specific promoter to target a specific organ, tissue, graft, tumor or cell And further transfected with signals and stabilization sequences for subcellularly localized expression in cell organelles.

Thus, the dual paratope and polypeptides, immunoglobulin single variable domains and nanobodies of the present invention can be administered systemically, for example, orally, in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. It can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or blended directly with the patient's food. For oral therapeutic administration, the double paratope polypeptides of the present invention may be combined with one or more excipients and used in the form of ingestible tablets, oral tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like . The compositions and preparations should contain at least 0.1% of the inventive double-paratovenous polypeptide, immunoglobulin single variable domains or nanobodies. The proportion thereof in the compositions and preparations can, of course, vary and can conveniently be from about 2 to about 60% of the weight of the unit dosage form presented. The double paratope polypeptides of the present invention in such therapeutically useful compositions are intended to provide effective dosage levels.

Tablets, troches, pills, capsules and the like may also contain binders, excipients, disintegrants, lubricants and sweetening or flavoring agents, such as those mentioned on pages 143-144 of WO 08/020079. When the unit dosage form is a capsule, it may contain a liquid carrier, for example vegetable oil or polyethylene glycol, in addition to the above-mentioned materials. Various other materials may be present as coatings or to otherwise alter the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, or the like. Syrups or elixirs may contain the dual parathetic nanobodies and polypeptides of the present invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, dyes and flavors such as cherry or orange flavor. Of course, any substance used in the manufacture of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, the dual parathetic nanobodies, immunoglobulin single variable domains and polypeptides of the present invention may be included in delayed-release formulations and devices.

Formulations and formulations for oral administration may also be provided with enteric coatings that allow the constructs of the present invention to withstand the gastric environment and allow passage into the intestines. More generally, formulations and preparations for oral administration can be suitably formulated for delivery into any desired portion of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.

The dual paratope nano-bodies, immunoglobulin single variable domains and polypeptides of the present invention may also be administered intravenously or intraperitoneally by injection or injection as further described in pages 144 and 145 of WO 08/020079.

For topical administration, the double-paratovenous nanobodies, immunoglobulin single variable domains and polypeptides of the present invention can be applied in pure form, i.e., when liquid. However, it is generally desirable to administer to the skin as a composition or formulation in combination with a skin-acceptable carrier, which may be either solid or liquid, as further described on page 145 of WO 08/020079.

Generally, the concentration of the dual parathetic nanobodies, immunoglobulin single variable domains and polypeptides of the invention in a liquid composition such as lotion will be about 0.1-25 wt-%, preferably about 0.5-10 wt-%. The concentration in the semi-solid or solid composition, such as gel or powder, will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of double-paratovenous nanobodies, immunoglobulin single variable domains and polypeptides of the invention required for use in therapy will depend not only on the particular double-paratope nano-body or polypeptide selected but also on the route of administration, the nature of the condition being treated, Will vary depending on the condition, and will ultimately be determined according to the judgment of the clinician or clinician. In addition, the dose of the double-paratovenous nanobodies and polypeptides of the present invention varies depending on the target cell, tumor, tissue, implant, or organ.

The required dose may conveniently be presented as a single dose or divided doses administered at appropriate intervals, for example, two, three, four or more sub-doses per day. The sub-doses themselves can be further divided, for example, into many separate roughly spaced doses, for example, multiple inhalations from the bite or application of many bite into the eye.

The dosing regimen may include long-term, one-day treatment. "Long term" means a period of at least two weeks, preferably several weeks, months, or even years. Required modifications of the above dosage ranges can be determined using only routine experimentation, taking into account the teachings of the present invention (see Remington ' s Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA]). The dosage can also be adjusted by the individual physician in the event of any complication.

In yet another aspect thereof, the present invention provides a method of inhibiting CXCR2 signaling by administering an effective amount of a polypeptide or pharmaceutical composition according to the present invention, preferably a dual paratopoietic immunoglobulin single variable domain or nanobody according to the present invention, And to a method for treating a disease or condition accompanied by abnormal function of the disease. As discussed herein, CXCR2 signaling mediates inflammatory responses in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD) leading to the destruction of pulmonary parenchyma. Leukocyte migration, seen as an increased number in the lungs of patients suffering from COPD, is associated with CXCR2 on the surface of the cell that binds to some ligands, including IL-8, Gro-α, β, γ, EMA 78 and GCP- Lt; / RTI &gt; The increase in the number of neutrophils in the lung is related to the severity of the disease. In addition, the concentration of Gro-alpha is significantly increased in induced sputum and bronchial washing (BAL) solutions of COPD patients. Thus, CXCR2 antagonism is expected to prevent, treat or alleviate the painful symptoms of the disease.

Accordingly, the present invention relates to a method for the prevention or treatment of COPD or COPD aggravation, comprising administering a double-paratopoietic polypeptide, such as a dual paratopoietic immunoglobulin single variable domain or nano-body of the invention, will be. The present invention also relates to the use of said double-paratovenous polypeptides comprising a double-paratovenous nanobody and a composition containing it, for the treatment of COPD or COPD aggravation.

The dual paratopoietic polypeptides of the present invention, particularly the dual paratoptic immunoglobulin single variable domains or nanobodies and compositions thereof, are also useful for the treatment of other conditions associated with abnormal function of CXCR2 signaling, such as other conditions of the respiratory tract, It will be readily apparent to those skilled in the art that useful for the treatment of cystic fibrosis, severe asthma, aggravation of asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, airway remodeling, obstructive bronchiolitis syndrome or bronchopulmonary dysplasia .

Additional diseases and conditions that can be prevented or treated by the dual paratopoietic polypeptides of the present invention, e. G., The dual paratopoietic immunoglobulin single variable domains or nanobodies and pharmaceutical compositions thereof, include atherosclerosis, glomerulonephritis, Diseases characterized by neovascularization including inflammatory bowel disease (Crohn's disease), angiogenesis, and macular degeneration, diabetic retinopathy and diabetic neuropathy, multiple sclerosis, psoriasis, age-related macular degeneration diseases, Chronic inflammatory disease, rheumatoid arthritis, osteoarthritis, non-small cell carcinoma, colon cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer, solid tumors and osteoarthritis, including osteoporosis, uveitis, idiopathic pulmonary arterial hypertension (PAH), familial PAH and related PAH Metastasis, melanoma, hepatocellular carcinoma, or ischemic reperfusion injury.

Additional diseases and conditions that can be prevented or treated by the double-paratovenous polypeptides of the present invention, e. G., The double-paratovenous immunoglobulin single variable domains or nanobodies and pharmaceutical compositions thereof, include hemolytic transfusion induction in sickle cell disease Vascular occlusion, ischemia / reperfusion injury, acute stroke / myocardial infarction, obstructive head injury, post-traumatic inflammation, and insulin-resistant diabetes mellitus.

For this method, the double-paratovenous nanobodies, immunoglobulin single variable domains and / or polypeptides and / or compositions comprising the same of the present invention may be administered in any suitable manner depending on the particular pharmaceutical formulation or composition used . Thus, the dual parathetic nanobodies and / or polypeptides and / or compositions comprising them of the present invention can be administered orally, intraperitoneally (e. G., Intravenously, subcutaneously, intramuscularly, Intravenous, intravenous, intraperitoneal, intravenous, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, Generally for COPD, inhalation is not a desirable route. The clinician will be able to choose the appropriate route of administration and the appropriate pharmaceutical formulation or composition to be used in the administration according to the needs of the individual patient.

The double paratope nano-bodies, immunoglobulin single variable domains and / or polypeptides and / or compositions comprising the same of the present invention are administered in accordance with a therapeutic regimen suitable for the prevention and / or treatment of a disease or disorder to be prevented or treated. The clinician will generally assess whether the disease or disorder to be prevented or treated, the severity of the disease being treated and / or the severity of the symptoms, the particular dual-paratope nano-body, immunoglobulin single variable domain or polypeptide of the present invention employed, The route of administration and the pharmaceutical agent or composition, the age, sex, weight, diet, general condition of the patient, and similar factors well known to those of skill in the art.

In general, for the prevention and / or treatment of diseases and disorders referred to herein, particularly COPD, the amount administered will depend upon a variety of factors including the potency of the particular dual-paratope nano-body, immunoglobulin single variable domain or polypeptide of the invention, Will depend upon the particular pharmaceutical formulation or composition employed. Generally, a dose of 1 gram to 0.01 microgram / kg (body weight) / day, preferably 0.1 gram to 0.1 microgram / kg (body weight) / day, such as about 1, 10, 100 or 1000 micrograms / / Day, continuously (e. G., By injection), as a single daily dose or as multiple subdoses for a day. The clinician will generally be able to determine the appropriate daily dose according to the factors mentioned herein. It will also be apparent that in certain instances, the clinician can select an altered amount from the amount based on, for example, the above-mentioned factors and their expert judgment.

The dual parathetic nano-bodies, immunoglobulin single variable domains and polypeptides of the present invention may also be used in combination with one or more additional pharmaceutical active compounds or ingredients, i. E. As combination therapies, which may or may not induce synergistic effects . Again, the clinician will be able to select such additional compounds or ingredients, and suitable combination therapies, based on, for example, the above-mentioned factors and their expert judgment.

For example, the dual paratopoietic polypeptides of the present invention, for example, the bileat paratovenous nano-body, can be administered to a subject in need of conventional therapies for COPD, such as short- and long-lasting? -Adrenergic bronchodilators, inhaled anticholinergics (muscarinic antagonists) It would be possible to combine it with a corticosteroid.

The effects of the therapeutic regimens used in accordance with the present invention can be determined and / or tracked in any manner known per se for the relevant disease or disorder, as will be apparent to the clinician. The clinician may also determine, in appropriate cases and on a case by case basis, to achieve the desired therapeutic effect, to prevent, limit or reduce unwanted side effects, and / or to achieve the desired therapeutic effect on the one hand, Or &lt; RTI ID = 0.0 &gt; inhibiting, &lt; / RTI &gt;

In general, the therapeutic regimen will be performed as long as the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect is to be maintained. Again, this can be decided by the clinician.

The subject to be treated may be any warm-blooded animal, but in particular a mammal, more particularly a human. As will be apparent to those skilled in the art, the subject to be treated will be a person who is afflicted with or at risk for the diseases and disorders referred to herein.

The invention will now be further illustrated by the following non-limiting preferred aspects, embodiments and drawings.

All publications mentioned herein are incorporated herein by reference.

Deposited Information

Six deposits of plasmid DNA with insert encoding sequence-optimized nanobody polypeptides presented in Table 32 were purchased from Novartis Pharma AG, Switzerland on June 15, 2010 under the name DSMZ (Deutsche Sammlung von Mikroorganismen and Zallkulturem GmbH, Inhoffenstrasse, 7B, D-38124, Braunschweig, Germany). The deposit was made in accordance with the Budapest Treaty on the Recognition of Microorganisms for the Purposes of Patent Procedure for the Worldwide Recognition of Microorganism Deposit for the purposes of patent on 28 April 1997, Lt; / RTI &gt;

1. Human and Sino CXCR2 Cloning

pcDNA3.1 (+) (Invitrogen, V790-20) is designed for high-level constitutive expression in a variety of mammalian cell lines. This includes the human cytomegalovirus early promoter, the bovine growth hormone (BGH) polyadenylation signal, the neomycin selection marker for mammalian cells, and this. Ampicillin resistance gene for selection in E. coli.

pVAX1 (Invitrogen, V260-20) is a plasmid vector designed for DNA vaccination. This includes the human cytomegalovirus early promoter, bovine growth hormone (BGH) polyadenylation signal, Lt; RTI ID = 0.0 &gt; kanamycin &lt; / RTI &gt; resistant gene for selection in E. coli.

Buildings : Receptor vector build Human CXCR2
(N-terminal 3 x HA-tag) pcDNA4 / TO Following the DNA sequence coding for the three HA tags, the huCXCR2 sequence was subcloned and cloned into the HindIII and XhoI restriction sites at the 5 'and 3' ends, respectively, in pcDNA4 / TO Human N-terminal CCR9 chimeric CXCR2
(N-terminal 3 x HA-tag) pcDNA4 / TO Following the DNA sequence coding for the three HA tags, the first 39 amino acids, the TEV protease site and the huCXCR2 sequence-N-terminal 43 amino acids for huCCR9 were subcloned and cloned at the 5 'and 3' ends in pcDNA4 / HindIII and XhoI restriction sites Human Δ1-17 CXCR2
(N-terminal 3 x HA-tag) pcDNA4 / TO Following the DNA sequence coding for the three HA tags, the huCXCR2 sequence lacking the N-terminal 17 amino acids was subcloned and cloned into the HindIII and XhoI restriction sites at the 5 'and 3' ends, respectively, in pcDNA4 / TO Human CXCR2 pXoon Human CXCR2 ( _h CXCR2) cDNA (GENBANK: L19593) was amplified by PCR using a 5 'primer containing an EcoRI cleavage site and a 3' primer containing a NotI site. The PCR product was ligated into the pXOON plasmid vector. Cynomolgus CXCR2 pcDNA3.1 Cynomolgus CXCR2 cDNA was amplified from the spleen / thymus cynomolgus cDNA library. The NotI and XhoI restriction enzyme sites were added via PCR and the resulting fragments cloned into pcDNA3.1. Human CXCR2 pVAX1 PCR for pXoon_hCXCR2 (NheI-NotI) Cynomolgus CXCR2 pVAX1 From pcDNA3.1_cCXCR2, NheI-XhoI Human Δ1-17 CXCR2 pVAX1 PCR (HindIII-XhoI) for pcDNA3.1_3x-HA-Δ1-17-hCXCR2 Human Δ1-17 CXCR2
(N-terminal 3 x HA-tag) pcDNA3.1 pCR4Blunt-TOPO_3xHA-Δ1-17-hCXCR2 to HindIII-XhoI Human CXCR2 pcDNA3.1 pVAX1_hCXCR2 to NheI-XhoI

2. Human and Cynomolgus CXCR2 Expressing CHO , CaKi , RBL  And HEK293T  Establishment of cell line

CHO - K1  ? 1-17 human CXCR2  (N-terminal 3 xHA  tag)

CHO-K1 cells were transfected with the plasmid pcDNA3.1_3xHA-Δ1-17-hCXCR2 using the Amaxa electroporation system (program U 23 in solution T). A pool of transfected cells was maintained from day 2 post-transfection under selective pressure (1000 [mu] g / mL G418). After 8 days, the human CXCR2 positive population was identified using FMAT Blue-labeled human Gro-α. The FMAT Blue label (BioSource, PHC1063) of human Gro-α was made using the FMAT Blue mono-functional reactive dye kit according to the manufacturer's instructions (Applied Biosystems, 4328408). Single cells were sorted into 96-well cell culture plates using FACSaria (BD Biosciences). Growing clones were tested for? 1-17 human CXCR2 expression on a FACSarray (vivo biosciences) device using FMAT Blue-labeled human Gro-α. CHO-K1 clones with the highest expression were selected (MCF value of 9000).

HEK293T Cynomolgus CXCR2

HEK293T cells were transfected with plasmid pcDNA3.1_cCXCR2 using FuGene HD transfection reagent (Roche). Two days after transfection, cells were tested for cCXCR2 expression on a FACSarray (vivo biosciences) device using 50 nM FMAT Blue labeled Gro-a. Cells with good expression (MCF values of about 12000) were used additionally.

RBL -2 H3 Cynomolgus CXCR2

Rat basophil leukemia cells (RBL-2H3) (MEM Eagle, grown at 37 占 폚 / 5% CO ₂ and supplemented with 1X non-essential amino acid, 0.15% sodium bicarbonate, 1 mM sodium pyruvate and 15% (Typically subcultured in Eagle's medium (Invitrogen)) were subjected to nucleofection by electroporation (Amaxa Biosystems) according to the manufacturer's protocol. Transfected cells were incubated at 37 ° C / 5% CO ₂ and 24 hours after transfection, antibiotic selection was initiated by adding geneticin to a final concentration of 1 mg / ml. Transfected cells were single-cell sorted by growing, subculturing for 3-5 days in selective medium, and serially diluting into 96-well plates. Approximately two weeks later, the actively growing colonies were expanded and subsequently analyzed for syno CXCR2 transcript expression. The positive clones were then further expanded for analysis.

CHO - Trex ( HA ) 3- huCXCR2  And ( HA ) 3 huCCR9 - CXCR2 hybrid  ( hybrid )

(FBS) (Biosera), 1% penicillin / streptomycin & lO &amp; 10 at 37 ° C in a Chinese Hamster Ovary T-Rex (T-Rex ™ -CHO, Invitrogen, # R718-07) and maintained in a monolayer culture medium in Ham's F12 medium containing 2 mM L-glutamine supplemented with μg / mL of blasticidin. The tetracycline-regulated expression (T-Rex ™) cell line stably expresses a tetracycline repressor (TetR). Subsequently, stable cell lines expressing both CXCR2 constructs were produced using a nuclear transfection procedure (cell line nuclear transfection kit T, AMAKA BIO SYSTEM, program U-23). Transfected cells were incubated at 37 [deg.] C / 5% CO ₂ and treated with 300 [mu] g / ml Zeocin 48 h after transfection. Cells were cultured for 2 weeks in the presence of eicosin to permit selection of positive transformants, after which single-cell sorting was performed using the Mo-Flo FACS classifier. After 2 weeks, colonies growing actively were inflated while maintained within their defined media at a concentration of 300 ug / mL of myosin.

3. Human Gro -α, Cynomolgus Gro -α, human IL -8, human ENA -78

NVTS-IL-8, ENA-78, Cynomolgus Gro-alpha Ligand Commentary Source Human GROα Recombination BioSource (PHC 1063) Human IL-8 Recombination Novartis Vienna Human ENA-78 Recombination Peprotech ltd (300-22) Sino GROα Recombination ALMAC Sciences

4. Peptides

Peptides presenting different N-terminal and extracellular loop (EL) stretches of human and Cynomolgus CXCR2 were ordered from Bachem (Table 5). In peptides designated as "cyclic ", the first and last amino acids were replaced with cysteine residues, and the naturally occurring internal cysteine of the wild-type sequence was replaced with a leucine residue. These peptides have been cyclized through side-flanking cysteine residues.

5. Immunization

Three rabbits were immunized seven to nine times with mammalian cells expressing human CXCR2 and one rabbit was immunized six times with mammalian cells expressing cynomolgus CXCR2. Following this regimen, a cocktail of peptide-keyhole limpet hemocyanin (KLH) conjugate mixed in complete Freund's Adjuvant was administered four times, and the peptide was administered to a human and a synomol Extracellular loop numbers 2 and 3 of both GUS CXCR2 (see Table 5). Eight other lambs were immunized four to five times with DNA encoding human full-length CXCR2 or pXAX1-expressing? 1-17 CXCR2, followed by a single dose of mammalian cells expressing human full-length CXCR2. In addition, three llamas were immunized four times with DNA encoding Cynomolgus CXCR2 expressed from pVAX1, and then the mammalian cells expressing Cynomolgus CXCR2 were administered once. Immunological blood and lymph node samples were taken 4 and 8 days after each antigen challenge.

6. Building the Library

cDNA samples were prepared from total RNA preparations of immune blood and lymph node samples. Nucleotide sequences encoding the nanobodies were amplified from all llama cDNA samples immunized with human or cynomolgus CXCR2 in a one-step RT-PCR reaction using primers ABL051, ABL052 and ABL003. The primer sequences are shown in Table 6. A 700 bp amplicon amplified from the IgG2 and IgG3 cDNA in the sample was isolated from the gel and subsequently used as a template in a nested PCR reaction using the ABL050 primer containing the SfiI restriction site and the ABL003 primer. The PCR product was subsequently digested with (a naturally occurring in FR4 of the V _HH gene) SfiI and BstEII and, to Lai gated into the corresponding restriction sites of phagemid vector pAX50, Escher Escherichia coli (Escherichia After electroporation in E. coli TG-1, a library was obtained. pAX50 is an expression vector derived from pUC119 containing a LacZ promoter, a coliphage pIII protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and a gen3 leader sequence. With the Nanobody® coding sequence, the vector encoded the C-terminal c-myc tag and the (His) 6 tag. The phagemid vector allows the production of phage particles that express individual nanobodies as fusion proteins with the gene III product.

7. Select

The above-mentioned pAX50 nano-body library expressed on the surface of bacteriophage was selected using peptides, membrane extracts and whole cells expressing the CXCR2 epitope.

Selection using peptides was carried out using 0-1000 nM biotinylated peptides (see Table 1) captured on a Nutravidin-coated (Pierce, 31000) Maxisorp microtiter plate (Nunc, 430341) 5). &Lt; / RTI &gt; Alternatively, the phage library was incubated with 10 nM biotinylated peptide in solution followed by capture of the peptide-phage complex on streptavidin-coated Dynabead (Invitrogen, 112-06D). Blocking was performed using PBS supplemented with 1% casein. The phage prepared from the library was added and incubated for 1 hour (in PBS supplemented with 0.1% casein and 0.1% tween20). Unbound phage were washed off (using PBS supplemented with 0.05% tween20); Bound phages were eluted by adding trypsin (1 mg / ml in PBS) for 15 minutes. The second selection round was performed essentially as described above.

Selection using membranous extracts was carried out using a 50 ug / mL (total protein) membrane extract prepared from cells expressing human CXCR2 (Perkin Elmer, ES-145-M400UA and 6110524400UA) &Lt; / RTI &gt; As a negative control, membrane extracts prepared from CHO cells expressing human FPR1 (PerkinElmer, 6110527400UA) were coated in parallel. Blocking was performed using PBS supplemented with 4% Marvel skimmed milk powder. The phage were incubated for 2 hours (in PBS supplemented with 1% marbella skim milk). Unbound phage were washed away with PBS; Bound phages were eluted by adding trypsin (1 mg / ml in PBS) for 15 minutes. The second round selection was performed essentially as described above. In some cases, a phage binding to an unrelated cell background epitope was specifically depleted by pre-absorbing the phage onto successive tubes or wells coated with a control membrane extract. The incubation on the coated human CXCR2 membrane extract was then carried out in solution in the presence of the control membrane extract. In another experiment, selection of one or two rounds of peptide followed by one round of selection of membrane extract was performed or vice versa.

In another set of experiments, 1-5 million mammalian cells expressing human or synomolgus CXCR2 were incubated with phage library in PBS supplemented with 10% FBS and 1% Marbel skimmed milk powder. Untransformed cell lines were used as negative control. Unbound phage were washed away with PBS; Bound phages were eluted by adding trypsin (1 mg / ml in PBS) for 15 minutes. The second round was performed essentially as described above, but for a different cell line background than the first round.

In another experiment, a mammalian cell or membrane extract expressing CXCR2 in the presence of 1 [mu] M of peptide (see Table 5) in solution to deplete the phage expressing the nanobody binding the phage to the region indicated by the peptide Lt; / RTI &gt;

8. Preparation of peripheral cytoplasmic extract

TG-1 cells exponentially growing with eluted phages were infected and then plated on carbenicillin-containing LB agar plates. Carbenicillin-resistant clones were analyzed for the presence of inserts and the sequence of positive clones was confirmed. Interesting clones were grown in TB medium supplemented with carbenicillin and expression was induced by addition of IPTG. After continued expression at 37 DEG C for 4 hours, the cells were centrifuged. this. Cell pellets frozen overnight from the coli expression broth were dissolved in PBS (1/10 of the original culture volume) and incubated for 1 hour at 4 ° C under gentle shaking conditions. The cells were then centrifuged one more time and the supernatant containing the secreted proteins into the surrounding cytoplasmic space was stored.

9. Screening

Peripheral cytoplasmic extracts (as described above) were analyzed for competition with Gro-α for binding to human CXCR2 on FACS. 2x10 ⁵ cells were incubated with a 1/2 dilution of the peripheral cytoplasmic extract in a FACS buffer (PBS + 10% TaqO 2 serum (Sigma, F7524) for 30 minutes at 4 ° C. The same volume of 6 nM of FMAT Blue-labeled human Gro-a in FACS buffer was then added and incubated further in the dark at 4 DEG C for 30 minutes. Cells were then washed three times in FACS buffer and finally resuspended in FACS buffer. Dead cells were stained with propidium iodide (Sigma, P4170). Samples were then analyzed on a FACSarray (Vidi Biosciences). Table 7 lists nanobodies whose peripheral cytoplasmic extracts compete with Gro-a for human CXCR2.

In another arrangement, peripheral cytoplasmic extracts were analyzed for binding to human 1-19 peptides by ELISA. MaxiSorb plates (Knock, 430341) were coated with neutravidin for 2 hours and then blocked for 1 hour (PBS, 1% casein). Subsequently, 100 nM biotinylated human 1-19 peptide was added to these plates for 1 hour (PBS, 0.1% casein, 0.05% tween 20) and incubated for 1 hour with a 10x dilution of peripheral cytoplasmic extract. Unbound unbound cytoplasmic extracts were washed away (PBS supplemented with 0.05% tween20) and the combined nanobodies were incubated with rabbit anti-mouse-HRP conjugate (Dakocytomation) following mouse anti-myc (Roche, 11667149001) , P0260). Table 8 summarizes the ratio of the combined signal of anti-CXCR2 nanobodies to that of non-related control nanobodies.

10. Sequence

1 Nano-body  Leading to characterization

11. One Nano-body  build

Nanobody-containing DNA fragments (Table 1) obtained by PCR on functional phagemid clones using Fwd-EVQL-MfeI and Rev-TVSS-BstEII primers were digested with MfeI and BstEII, ligated into pAX100 vector, this. Lt; RTI ID = 0.0 &gt; TG-1 &lt; / RTI &gt; competent cells. pAX100 is an expression vector derived from pUC119 containing a LacZ promoter, a resistance gene for kanamycin, a multicloning site, and an OmpA leader sequence. Together with the nanobody coding sequence, the vector encoded the C-terminal c-myc tag and the His6 tag. A kanamycin resistant clone was analyzed for the presence of the insert and the sequence of the positive clone was confirmed.

12. Small expression

TG-1 cells containing the expression vector coding for the nano-body of interest were grown in baffled shaker flasks containing TB medium + 100 [mu] g / ml kanamycin and 1 mM IPTG was added to induce expression Respectively. Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; After collection of the cells, peripheral cytoplasmic extracts were prepared and the His6-tagged nanobodies were desalted in PBS (HiPrep 26 / sup>) in PBS followed by fixed metal affinity chromatography (HisTrap FF Crude, GE Healthcare) 10, Zihealthcare) or gel filtration chromatography (Superdex 75 HR16 / 10, Zihealthcare).

13. Ligand  Competition test

The purified monovalent anti-CXCR2 nanobodies were titrated against 3 nM FMAT-Blue-labeled Gro-a by FACS ligand competition assay against human and Cynomolgus CXCR2 (Table 10). The blocking effect for human CXCR2 is in the range of two digits to less than nM, while for the Cynomolgus CXCR2 is in the range of one to two digits nM.

14. Functional assays using recombinant cell lines

(One) Intracellular  Of calcium Efficacy agent  Measurement of induced emissions ( FLIPR )

RBL cells expressing human or synomolgus CXCR2 receptor were inoculated into 96-well plates and incubated overnight at 37 &lt; 0 &gt; C. On the day of the experiment, the cells were loaded with Fluo-4 dye for 30 min at 37 [deg.] C and then incubated for 30 min with the purified monovalent anti-CXCR2 nanobodies. Finally, the addition of Gro-alpha was performed using a Fluorescence Imaging Plate Reader (FLIPR) and fluorescence signals corresponding to the release of intracellular calcium were detected. The selectivity test was performed using L2071 cells expressing human CXCR1. The assay protocol remained the same as described for CXCR2, but IL-8 was used as an agonist. A summary of the mean IC ₅₀ values is shown in Table 11 and further, any of the nanobodies tested showed any inhibition of the potentiator-induced release of intracellular calcium at the CXCR1 receptor at the concentration tested (1 μM maximum concentration) I did.

(2) [ ³⁵ S] GTP of γS Efficacy agent  Measurement of stimulated accumulation

The purified monovalent anti-CXCR2 nanobodies were incubated for 60 minutes with CHO-CXC2 membranes prepared from CHO cells expressing Gro-a, GDP, SPA beads and human CXCR2 receptor in 96-well plates. Subsequently, [ ³⁵ S] GTPγS was added and incubated for an additional 60 minutes. Finally, the plates were centrifuged and read on a Topcount. A summary of the average IC ₅₀ values is presented in Table 11.

15. Functional assays using primary neutrophils

(1) Human neutrophil whole blood shape change test hWBSC )

The donor was a healthy normal volunteer who did not take the whole body medication (Novartis Horsham donor panel). Whole blood anticoagulated with 52 mM EDTA (sterilized) was collected with a ratio of 1 mL EDTA to 9 mL blood. Blood was collected at room temperature and pre-warmed to 37 캜 before use. After incubation (10 dots per dose response (0.03-1.144 x 10 ^-7 μΜ), were stimulated with a chemokine-a whole blood of 80 ㎕ spare at room temperature for 10 min with CXCR2 Nanobodies; 10 ㎕ rhGROα (2 nM Approximate EC ₇₀ concentration) was added to all wells except zero compound with 10 μL shape change assay buffer. Samples were gently shaken and incubated for an additional 5 minutes at 37 ° C. The tubes were then washed with ice , 250 μL of ice-cold optimized CellFix ™ solution was added to the gently shaken tube and incubated for an additional 5 minutes, then 1.4 mL of 1 × ammonium chloride lysis solution was added to all tubes and added on ice (Becton Dickinson) After the red cell dissolution, the sample was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) .The cell population was analyzed with a scatter / side scattering probe (FSC / SSC) gating followed by FSC / FL-2 plots using gated granulocytes from the first plot. Neutrophils were distinguished from eosinophils on the FL-2 plot, 5000 events per sample were counted.

(2) human neutrophils Chemistry  black

The donor was a healthy normal volunteer who did not take the whole body medication (Novartis Hotham donor panel). Whole blood anticoagulated with 52 mM EDTA (sterilized) was collected with a ratio of 1 mL EDTA to 9 mL blood. Leukocytes were isolated using standard protocols: 4% dextran was added to 20 mL anticoagulated blood, gently mixed, and incubated on ice for 30 min to precipitate red blood cells. The supernatant containing peripheral blood mononuclear cells (PMN) was then laminated onto a Ficoll-Paque (R) density gradient and centrifuged at 300 xg for 25 min at 18 [deg.] C. The PMN rich fraction was resuspended in 500 [mu] l 1X PBS and red cell dissolution was performed using a shelf-life shock. 20 mL ice-cold sterile endotoxin-free distilled water was added to the pellet and allowed to dissolve for 30-40 seconds before adding 20 mL 2X PBS. The samples were gently mixed and centrifuged at 300 x g for 10 minutes at 18 DEG C to obtain granules. The granulocyte pellet was resuspended in 500 l Ix PBS and washed twice with 50 ml xl PBS. Resuspended, and counted the granulocyte pellet in RPMI 1640 (pH 7.4) + 2.5 % FBS , and diluted to a final concentration of 2e ⁶ / mL. Transfer was measured using a transwell plate with a 3 占 퐉 PET membrane (Becton Dickinson). Briefly, 6 nM of GRO alpha (EC ₈₀ -EC ₁₀₀ ) was added to the lower well of the plate (1000 μl / well), the multiwell insert was lowered to the position and then the variable concentration nanobodies (500 [mu] l / well) pre-incubated PMN at RT for 30 minutes with 0.13-1000 [mu] M for double-stranded or 0.6 [ Plates were then incubated at 37 ° C for 90 minutes and the cells transferred to the lower chamber were counted using a FACSCalibur flow cytometer. The flow cytometer was set to count the number of events in the R2 gate on the FSC / FL-2 plot for a set time of 20 seconds per sample.

(3) Cynomolgus  Neutrophil whole blood shape change test WBSC )

Venous blood from the forearm or leg, anticoagulated with 3.8% sodium citrate (sterilized), was added at a ratio of 1 mL of sodium citrate to 9 mL of blood. Blood was collected at room temperature and pre-warmed to 37 캜 before use. After incubation (10 dots per dose response (0.03-1.144 x 10 ^-7 μΜ), were stimulated with a chemokine-a whole blood of 80 ㎕ spare at room temperature for 10 min with CXCR2 Nanobodies; 10 ㎕ rhGROα (30 nM approximate EC ₇₀ concentration of _-90) for 10 ㎕ shape changes are added to all wells excluding the buffer test compound was added to a zero. gently shake the sample, which was added to incubate at 37 ℃ for 5 minutes. then, the tube of ice , 250 μl of ice-cold optimized CellFix ™ solution was added to the gently shaken tube, incubated for an additional 5 minutes and then 2 mL of dissolution buffer (Sigma Aldrich # R7757) was added to all tubes The samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson). After collecting the cell population with a front scattering detector / side scattering detector Following gating (FSC / SSC), gating was confirmed by FSC / FL-2 plots using gated granulocytes from the first plot. Neutrophils were distinguished from eosinophils on the FL-2 plot, 5000 events per sample were counted.

Close Nanobody

16. Two Nano-body  build

Two approaches were used to build a 2-nanobody body.

PCR amplification was performed on the plasmid DNA encoding a monovalent building block. The N-terminal building block was amplified using a reverse primer coding for Fwd-EVQL-MfeI and a portion of the GlySer linker while the C-terminal building block was amplified using forward primer coding for the remainder of the GlySer linker and Rev-TVSS-BstEII (Table 6). The N-terminal fragment is digested with MfeI and BamHI, the C-terminal fragment is digested with BamHI and BstII; They are ligated simultaneously into the pAX100 vector, Lt; RTI ID = 0.0 &gt; TG-1 &lt; / RTI &gt;

Alternatively, different PCR amplifications were performed on plasmid DNA encoding univalent building blocks. The N-terminal building blocks were amplified using Fwd-EVQL-MfeI and Rev-TVSS-BspEI, while the C-terminal building blocks were amplified using Fwd-EVQL-BamHI and Rev-TVSS-BstEII (Table 6) . The N-terminal fragment was digested with MfeI and BamHI, and the C-terminal fragment was digested with BspEI and BstII. The N-terminal fragment was ligated into a pAX100-derivative containing coding information for the GlySer linker (MfeI-BspEI) Lt; RTI ID = 0.0 &gt; TG-1 &lt; / RTI &gt; Plasmid DNA from the transfected mixture was prepared and digested with BspEI and BstEII, followed by ligating the C-terminal fragment into the pAX100 vector. Lt; RTI ID = 0.0 &gt; TG-1 &lt; / RTI &gt;

A kanamycin resistant clone was analyzed for the presence of the insert and the sequence of the positive clone was confirmed.

17. Multi- CXCR2 Nano-body  order

18. Ligand  Competition test

The polyvalent anti-CXCR2 nanobodies were titrated against 3 nM FMAT-Blue-labeled Gro-a with FACS ligand competition assay against human and Cynomolgus CXCR2 (Table 14). The blocking effect for human CXCR2 is in the range of two digits to less than nM, while for the Cynomolgus CXCR2 is in the range of one to two digits nM.

19. Functional assays using recombinant cell lines

(One) Intracellular  Of calcium Efficacy agent  Measurement of induced emissions ( FLIPR )

RBL cells expressing human or synomolgus CXCR2 receptor were inoculated into 96-well plates and incubated overnight at 37 &lt; 0 &gt; C. On the day of the experiment, the cells were loaded with Fluo-4 dye for 30 min at 37 [deg.] C and then incubated for 30 min with the purified polyvalent anti-CXCR2 nanobody. Finally, the addition of Gro-alpha was performed using a Fluorescence Imaging Plate Reader (FLIPR) and fluorescence signals corresponding to the release of intracellular calcium were detected. The selectivity test was performed using L2071 cells expressing human CXCR1 using IL-8 as an agonist and CX cells expressing human CXCR4 endogenously using SDF-1 as an agonist, The assay protocol remained the same as described for CXCR2. A summary of the mean IC ₅₀ values is presented in Table 15 and additionally any inhibition of the agonist-induced release of intracellular calcium in CXCR1 or CXCR4 at any of the tested nanobodies tested concentrations (1 μM maximum concentration) I did not see it.

(2) [ ³⁵ S] GTP of γS Efficacy agent  Measurement of stimulated accumulation

The purified polyclonal anti-CXCR2 nanobodies were transfected into CHO-CXC2 membranes prepared from CHO cells expressing agonists (GRO-alpha, IL-8 or ENA-78) GDP, SPA beads and human CXCR2 receptor in 96- &Lt; / RTI &gt; for 60 minutes. Subsequently, [ ³⁵ S] GTPγS was added and incubated for an additional 60 minutes. Finally, the plates were centrifuged and read on top counts. A summary of the average IC ₅₀ values is presented in Table 15.

(3) The anti- CXCR2 Nano-body  Action Mechanism  To decide Shilton  ( Schild ) analysis

IL-8 and Gro-alpha stimulated [&lt; ³⁵ &gt; S] GTPyS accumulation assays. The assay format allowed equilibrium of agonists and nanobodies prior to the addition of [ ³⁵ S] GTPγS and as a result, any artifacts of half-equilibrium that could lead to misinterpretation of the mechanism could be avoided. To this end, an agonist concentration response curve was determined in the presence of increasing concentrations of nanobodies. The data for the two 1D nanobodies 54B12 and 163E3 and the resulting multibranched nanobodies are given as an example. The data show concentration response curves for Gro-α, but similar data were obtained using IL-8.

Both monovalent nanobodies 54B12 and 163E3 exhibit an allosteric mechanism of action, but have a differential effect on the inhibition of agonists. The Alrotic mechanism of 54B12 and other 1-19 binders is illustrated by the parallel rightward movement of the agonist concentration response curve at low nanobody concentrations that do not migrate further to the right in the presence of increasing concentrations of nanobodies. The saturatable properties of the effect without exhibiting a decrease in maximal efficacy response indicate the effect of alosteri on the affinity of the agonist. In contrast, the allo-terim mechanism of 163E3 and other non-1-19 binders is exemplified by the parallel rightward movement of the agonist concentration response curve in combination with the reduction of maximal agonist response at higher nanobody concentrations. Although this effect is saturable, it has not been observed at the concentrations used, however, the key observation is the reduction of maximal efficacy response indicating the effect of the agonist on the efficacy of the agonist. Finally, the multivalent nanobodies 54B12-163E3 were used to generate effects on the agonist concentration response curve as illustrated by a parallel right shift at much lower nanobody concentrations and a significant reduction in maximum agonist response, Mechanisms are combined.

The current definition of an alloosteric modulator is that it binds at a site that is separate from the agonist (orthosteric ligand) binding site and that both the orthosteric ligand and the allosteric modulator bind simultaneously to the receptor It is. The present inventors do not currently have the data to confirm this and do not wish to depend on the theory, but it is considered that the nano-body binding site is not distinguished from the effector binding site and the binding site is overlapped. In addition, data showing that both agonists and nanobodies are bound to the receptor at the same time is not available, but Schillt analysis data suggests that these nanobodies are the allosteric modulators of CXCR2.

20. Functional Test - NSC

Same method as described in Section 15

Lead panel (leadpanel) -CDR + FR CXCR2 Kabat

21. Sequence Optimization - CXCR2  Antagonist polypeptide

Thermal Conversion Assay ( TSA ): 5 μl of purified monovalent nanobodies (80 mg / ml) were mixed with 10 μl of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, 115 mM NaCl, (Invitrogen, Carlsbad, Calif., catalog # S6551) (final concentration 10x) with 5 μl of fluorescent probe ciprofloxacin (buffered at pH). The sample was then heated from 37 to 90 占 폚 at 4.4 占 폚 / s in a LightCycler 480II instrument (Roche, Basel, Switzerland) and then cooled to 37 占 폚 at 2.2 占 폚 / s. After heat-induced unfolding, the hydrophobic patch of protein is exposed and the orange to the cypher binds to it to increase the fluorescence intensity. The inflection point of the first derivative of the fluorescence intensity curve serves as a measure of the melting temperature (Tm) (Ericsson et al., 2006 (Annals of Biochemistry, 357: 289-298)).

Differential Scanning Calorimetry ( DSC ): The experiments were performed with Auto-Cap VP-DSC (MicroCal - GE Healthcare) according to the manufacturer's instructions. The melting temperature determination of the nanobodies (0.25 mg / ml) was carried out at a heating rate of 1 占 폚 / min over the temperature range of 30 占 폚 to 95 占 폚. The final thermogram was obtained after appropriate baseline subtraction. Peak detection through software (Origin 7.0) resulted in a corresponding melting temperature.

Forced oxidation: Nanobodies sample (1 mg / ml) is then applied at RT in 10 mM H ₂ O ₂ in PBS in combination with a control sample with no H ₂ O ₂ and cow for 4 hours, Zeba desalting spin column (0.5 ml) (Thermo Scientific) was used to convert the buffer to PBS. Stressed and control samples were then analyzed by RPC on a Zorbax 300SB-C3 column (Agilent Technologies) at 70 ° C with a Series 1200 instrument (Agilent Technologies). Oxidation of the nanobodies was quantified by determination of the% peak area of the pre-peak resulting from oxidative stress as compared to the main protein peak.

2B2 sequence optimization

The protein sequence of Mo2B2 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 20, page 219). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 2B2, CXCR20059, and CXCR20063 and was then characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2 . In addition, the melting temperature of the mutants was determined by Thermal Conversion Assay (TSA) or Differential Scanning Calorimetry (DSC) (Table 21). The M93L mutation in CXCR20059 and CXCR20063 eliminates the susceptibility of Mo2B2 to forced oxidation.

97A9 sequence optimization

The protein sequence of parent 97A9 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 22, page 220). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 97A9 and CXCR20061 and subsequently characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2. In addition, the melting temperature of the mutants was determined by the Thermal Conversion Assay (TSA) (Table 23).

163 E3  Sequence optimization

The protein sequence of MoI 163E3 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 24, page 221). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 163E3 and CXCR20076 and subsequently characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2. In addition, the melting temperature of the mutants was determined by the Thermal Conversion Assay (TSA) (Table 25).

127 D1  Sequence optimization

The protein sequence of MoI 127D1 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 26, page 222). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 127D1 and CXCR20079 and subsequently characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2. In addition, the melting temperature of the mutants was determined by the Thermal Conversion Assay (TSA) (Table 27). The M57R mutation in CXCR20079 eliminates the susceptibility of the parent 127D1 to forced oxidation.

163 D2  Sequence optimization

The protein sequence of MoI 163D2 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 28, page 223). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 163D2 and CXCR20086 and subsequently characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2. In addition, the melting temperature of the mutants was determined by the Thermal Conversion Assay (TSA) (Table 29).

54B12 sequence optimization

The protein sequence of parent 54B12 was aligned to human VH3-23 (DP-47) and JH5 reproductive sequences (Table 30, page 224). The amino acid differences for human germline sequences are indicated by letters and the same amino acids by dots. The underlined amino acid differences were selected for conversion to human counterparts and the others were left untouched.

The purified monovalent material was produced from 54B12, CXCR20103 and CXCR2104 and was then characterized in an effector-induced release (FLIPR) assay of intracellular calcium against both the FACS ligand competition assay and human and Cynomolgus CXCR2 . In addition, the melting temperature of the mutants was determined by the Thermal Conversion Assay (TSA) (Table 31).

22. Epitope Mapping  ( mapping )

The epitope mapping of the nanobodies was performed using the Shotgun mutagenesis technique, using integral Molecular Ink. (Integral Molecular Inc., Philadelphia Suite 900 Market Street 3711, Pennsylvania, USA, www.integralmolecular.com).

Shotgun mutation induction technique summary

Shotgun mutagenesis utilizes a proprietary high throughput cell expression technique that allows the expression and analysis of large libraries of mutated target proteins in eukaryotic cells. All residues in the protein are mutated individually into a number of different amino acids to test for changes in function. Proteins are expressed in standard mammalian cell lines, and even difficult proteins that require eukaryotic translation or post-translational processing can be mapped.

Nomenclature for epitope mapping is as follows:

RDHBC 792 = CXCR20079

RDHBC 793 = CXCR20061

RDHBC 792 = CXCR20076

The epitopes of the anti-CXCR2 antibodies RD-HBC792 (CXCR20079), RD-HBC793 (CXCR220061) and RD-HBC794 (CXCR20076) were mapped in single amino acid resolution using shotgun mutagenesis as follows.

The parental construct: the untagged parental gene was cloned into a high-expression vector, sequenced and verified for expression by immunodetection. Nanobody optimization: Detection of nanobodies was optimized in a shotgun mutagenesis format by assaying a series of nanobody dilutions in a 394-well microplate. The optimal concentration of each nanobody was selected to screen the mutant library. The mutation library was completed and the respective amino acid positions were mutated to conserved and non-conserved changes, including mutations in all residues to Ala substitutions. The library was tested for surface expression and triplicated for nano-body binding by immunodetection. Analysis of the library for loss of nanobody binding was performed, and important residues were identified and mapped.

Immunoassay Expression: Immunodetection of transiently expressed wild type embryo constructs was performed in 384 well format by immunoluminescence and immunofluorescence. For all experiments, liquid handling steps involving cell transfection and immunostaining were performed using liquid handling robots to ensure accuracy and high experimental reproducibility.

Experimental parameters used to test the parent plasmid Experimental parameter Immunoluminescence Immunofluorescence cell HEK-293T HEK-293T Fixer 4% PFA 4% PFA Blocking buffer 10% goat serum 10% goat serum 1 ° MAb target concentration
Incubation
Manufacturer Catalog # α-CXCR2 2 μg / ml for 1 hour R & D
Systems MAB331 α-CXCR2 3 μg / ml for 1 hour R & D
Systems MAB331 2 ° MAb target concentration
Manufacturer Catalog # α-mouse HRP 0.8 μg / ml
Jackson Immunori Search 115-035-003 alpha-mouse Dyelight 549 3.75 [mu] g / ml Jackson ImmunoResearch
115-505-003 wash PBS ++ PBS ++ Signal: Background 29: 1 2.2: 1 The CV% 4.7% 12%

Polyclonal  Experimental parameters used for immune detection Detection of total receptor cell surface expression using polyclonal serum. The polyclonal serum (which may react with all mutants) is used to quantify total expression so that each clone in the mutant library can be detected. Experimental parameter Polyclonal  Immunodetection cell HEK-293T Fixer 4% PFA Blocking buffer 10% goat serum 1 ° PAb target concentration
Incubation manufacturer catalog # α-CXCR2 1: 1,000 dilution 1 hour
Nobus NBP1-49218 2 ° MAb target concentration
Manufacturer Catalog # α-rabbit HRP 0.8 μg / ml
Southern Bio Tech 4050-05 wash PBS ++ Signal: Background 17: 1 % CV 10%

Conclusions: Strong surface expression and total expression are detected for wild-type parent constructs using control MAbs and polyclonal serum, and thus wild-type parent constructs can be used for shotgun mutagenesis. Immunogenicity assays show high signal: background and low variability and will be used for mapping studies.

Immune detection was optimized using a mapping nanobody. Immunodetection was performed in 384-well format using cells transiently transfected with wild-type receptor or vector-only plasmid. The concentrations selected for further mapping studies were based on signals close to the maximum signal with high signal: background and low variability.

Final assay conditions for screening of mutant libraries

shotgun  Experimental parameters used in the detection of optimized assays in mutagenic 384-well formats Experimental parameter RD - HBC792 RD - HBC793 RD - HBC794 cell HEK-293T HEK-293T HEK-293T Fixer 4% PFA 4% PFA 4% PFA Blocking buffer 10% goat serum 10% goat serum 10% goat serum 1 DEG MAb
Target
Optimum concentration
Incubation
α-CXCR2
1.0 / / ml
1 hours
α-CXCR2
1.0 / / ml
1 hours
α-CXCR2
2.0 / / ml
1 hours 2 ° MAb
Target concentration
Incubation
Manufacturer
Name of antibody
α-myc 2 μg / ml
1 hours
man
Hybridoma 9E10
α-myc 2 μg / ml
1 hours
man
Hybridoma 9E10
α-myc 2 μg / ml
1 hours
man
Hybridoma 9E10 3 ° MAb
Target
density
Manufacturer
catalogue #
α-mouse HRP
0.8 / / ml
Jackson Immunori Search
115-035-003
α-mouse HRP
0.8 / / ml
Jackson Immunori Search
115-035-003
α-mouse HRP
0.8 / / ml
Jackson Immunori Search
115-035-003 wash PBS ++ PBS ++ PBS ++ Signal: Background 13: 1 6.9: 1 20: 1 CV% 7.9% 22% 13%

The optimized assay conditions defined herein were used to map the CXCR2 mutant library in a 384-well format. Each clone in the library was expressed in cells by transient transfection and assayed for nanobody reactivity approximately 18 hours after transfection. The CXCR2 nanobodies RD-HBC792, RD-HBC793 and RD-HBC794 lacked the Fc region, but after using the multistage detection strategy containing the myc- tag and thus the intermediate mouse anti-myc antibody (9E10) Mouse HRP antibody.

Conclusion: The final conditions for immunodetection and epitope mapping of three CXCR2 nanobodies were determined. Optimized conditions produced high signal: background and low variability in the shotgun mutagenesis format and thus could be used for epitope mapping with high confidence. The epitope mapping entailed using the same assay conditions as determined here, but using a mutant library of receptor variants.

Nanobody On the epitope  important Residue  Confirm

Important residues in MAbs were identified by comparing the nanobody reactivity of the clones to the polyclonal reactivity (surface expression). The residues involved in the antibody epitope were negative for the nanobody binding but were identified as being positive for the polyclonal bond and contained an Ala residue substitution (i.e., removal of the side chain of the residue) and were located in the extracellular loop. Average reactivity and standard deviation for MAb binding and polyclonal antibody binding are presented. Significant residues identified for each MAb were shaded gray. In addition, data for RD HBC792 was compared to RD HBC793, because the binding profile for RD HBC792 was derived from commercial polyclonal serum (derived from the N-terminal extracellular domain of human CXCR2, probably F11, F14, and RTI ID = 0.0 &gt; W15 &lt; / RTI &gt; mutation).

Epitope  Additional analysis of information

Important amino acids identified by shotgun mutagenesis mapping define the binding site (s) for three CXCR2 MAbs. MAb RD HBC792 is mapped to the N-terminal region of CXCR2, and the close proximity of key residues suggests that the epitope is substantially linear. MAb RD HBC793 and RD HBC794 appear to bind primarily to the conformationally complex epitopes formed by ECLl and ECL3 of CXCR2. Also, a mutation of the extracellular Cys residue known to form two disulfide bridges that hold the extracellular loop in place in the chemokine receptors eliminates the binding of MAbs 793 and 794, and thus is directly involved in epitope interaction I do not think. Subtle differences between the two are apparent, but the epitopes 793 and 794 are significantly redundant.

Embodiment

1. A composition comprising a first antigen binding domain comprising at least two immunoglobulin antigen binding domains and acting against or binding to the chemokine receptor CXCR2 and recognizing a first epitope on CXCR2 and a second antigen recognizing a second epitope on CXCR2 A polypeptide comprising a binding domain.

2. The method of embodiment 1 wherein said first antigen binding domain is capable of binding to a linear peptide comprising the sequence of amino acids set forth in SEQ ID NO: 7 and wherein said second antigen binding domain is capable of binding to said linear peptide, Lt; / RTI &gt; polypeptide.

3. The method of embodiment 1 or 2 wherein said first antigen binding domain is comprised within a first immunoglobulin single variable domain and said second antigen binding domain is comprised within a second immunoglobulin single variable domain of an antibody / RTI &gt;

4. The polypeptide of embodiment 3, wherein at least one of the first and second antigen binding domains is comprised within the antibody V _L domain or fragment thereof.

5. The polypeptide of embodiment 3, wherein at least one of said first and second antigen binding domains is comprised within an antibody V _H domain or fragment thereof.

6. The polypeptide of embodiment 4 or 5, wherein said first antigen binding domain is comprised in a V _L domain or fragment thereof and said second antigen binding domain is comprised in a V _H domain or fragment thereof.

7. The polypeptide of embodiment 4 or 5, wherein said first antigen binding domain is comprised in a V _H domain or fragment thereof and said second antibody binding domain is comprised in a V _L domain or fragment thereof.

8. The polypeptide of any one of embodiments 3-7, wherein said first and second antigen binding domains are comprised within first and second domain antibodies (dAb).

9. The method of embodiment 3 or embodiment 5 wherein at least one of said first and second antigen binding domains is contained within a V _HH domain or fragment thereof from a single heavy chain of a heavy chain antibody obtainable from camelid, And a polypeptide that is sequence-optimized variant thereof.

10. The polypeptide of embodiment 9, wherein each said antigen binding domain is a sequence-optimized variant thereof, including in a V _HH domain or a fragment thereof from a single heavy chain of heavy chain antibodies derived from camelid, or humanized.

11. The polypeptide of embodiment 9 or 10, wherein each V _HH sequence or fragment thereof comprises one, two or three CDRs.

12. The polypeptide according to any one of embodiments 9-11, wherein each V _HH sequence has the structure of FR-CDR-FR-CDR-FR-CDR-FR.

13. The polypeptide according to any one of the preceding embodiments, wherein said at least two antigen binding domains are linked by a linker.

14. The polypeptide of any one of embodiments 3-13, wherein the polypeptide has the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8

Wherein FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 comprises a second antigen domain and FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 comprises a first antigen binding domain, When FR2-CDR2-FR3-CDR3-FR4 comprises a second antigen domain, FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 comprises a first antigen binding domain.

15. The polypeptide of embodiment 1 or 2, wherein each of said first and second antigen binding domains is comprised within a heavy chain antibody.

16. The method of embodiment 1 or 2 wherein said first antigen binding domain is comprised within a single chain of a first heavy chain antibody and said second antigen binding domain is comprised within a single chain of a second heavy chain antibody, And the second single heavy chain antibody is linked by a linker.

17. The polypeptide of embodiment 1 or 2, wherein each of said first and second antigen binding domains is comprised in an antibody consisting of two heavy chains and two light chains.

18. The antibody of embodiment 1 or 2, wherein said first and second antigen binding domains are each contained in a first and a second antibody, each comprising two heavy chains and two light chains, Wherein the polypeptide is linked by a linker.

19. The method of embodiment 1 or 2 wherein said first and second antigen binding domains are comprised within first and second antibody Fab or F (ab) 2 fragments, respectively, said first and second Fab or F (ab ) &Lt; / RTI &gt; fragment is linked by a linker.

20. The polypeptide of embodiment 19, wherein said first antigen binding domain is comprised within an antibody Fab fragment and said second antigen binding domain is comprised within an F (ab) 2 fragment or vice versa.

21. The method of embodiment 1 or 2, wherein said first and second antigen binding domains are comprised in first and second antibody single chain Fv (scFv) or fragments thereof, respectively, and said first and second scFv are linked to a linker &Lt; / RTI &gt;

22. In any one of embodiments 13, 14, 16, 18, 19, 20, or 21, the C-terminus of one immunoglobulin wherein the linker comprises an antigen binding domain is replaced with a second immunoglobulin Terminus of the polypeptide.

23. The polypeptide according to any one of embodiments 13, 14, 16, or 18 to 22, wherein the linker is a peptide comprising an amino acid sequence not derived from an immunoglobulin.

24. The polypeptide of embodiment 23 wherein the peptide linker is between 3 and 50 amino acids in length.

25. The method of embodiment 24 wherein the number of amino acids in the linker is selected from 3 to 9, 10 to 15, 16 to 20, 21 to 25, 26 to 35, 36 to 40, 41 to 45 or 46 to 50 Polypeptide.

26. The polypeptide of embodiment 24 wherein the linker is 35 amino acids in length.

27. The polypeptide of embodiment 24, wherein the linker consists of only two different amino acids.

28. The polypeptide according to embodiment 27, wherein the linker is composed of amino acid glycine and serine.

29. The polypeptide of embodiment 27 wherein the linker is made up of amino acid proline and serine and optionally alanine.

30. The polypeptide according to any one of embodiments 23-26, wherein the linker consists solely of alanine.

31. The polypeptide of embodiment 26, wherein the peptide linker is comprised of the amino acid sequence set forth in SEQ ID NO: 220.

32. The polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises a CDR amino acid sequence derived from or characterized by a camelid single-chain antibody.

33. The polypeptide according to any one of embodiments 9-14, wherein the amino acid sequence of the framework region is derived from or characterized by a camelid single chain antibody.

34. The polypeptide according to any one of embodiments 9-14, wherein the amino acid sequence of the framework region is humanized and the sequence-optimized variant thereof.

35. A polypeptide that specifically binds to CXCR2 in any one of the above embodiments.

36. In any one of the above embodiments, the polypeptide acts on or binds to human CXCR2.

37. The polypeptide of any of the preceding embodiments, comprising at least one additional antigen binding domain that specifically binds or acts on serum albumin.

38. The polypeptide of any one of the preceding embodiments, wherein the serum protein is human serum albumin.

39. A polypeptide comprising at least two polypeptides, wherein the first polypeptide comprises a first immunoglobulin antigen binding domain, the second polypeptide comprises a second immunoglobulin antigen binding domain, the first and second antigen binding domains Is a molecule that acts on or binds to the chemokine receptor CXCR2, which recognizes the first and second epitopes on CXCR2, and wherein said at least two polypeptides are linked by a non-peptide linker.

40. The method of embodiment 38, wherein said first antigen binding domain is capable of binding to a linear peptide consisting of the sequence of amino acids set forth in SEQ ID NO: 7, said second antigen binding domain not being capable of binding to said linear peptide, Molecule that is to merge road.

41. The molecule of embodiment 39 or 40, having the features defined in any one of embodiments 3-22 and 32-38.

42. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, And at least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the first immunoglobulin of the single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 161, and 181 or SEQ ID NO: , 161 or 181, and at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 181 (163D2 / 127D1).

43. The method of embodiment 42 comprising the structure set forth in embodiment 14 wherein CDR1 in the second immunoglobulin of said single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 145 and CDR2 has the amino acid sequence set forth in SEQ ID NO: 165 Wherein CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 185, CDR4 in the first immunoglobulin of said single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 161, and CDR6 Lt; RTI ID = 0.0 &gt; 181. &lt; / RTI &gt;

44. The method of embodiment 43 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 145, 165, 185, 141, 161 or 181 &Lt; / RTI &gt;

45. The polypeptide of embodiment 43 or 44, wherein the amino acid sequence differs only in conservative amino acid changes from those shown in SEQ ID NOs: 145, 165, 185, 141, 161 or 181.

46. The method according to any one of embodiments 43 to 45, wherein the framework region in each monomer of the single variable domain is at amino acid positions 11, 37, 44, 46, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

47. In any one of embodiments 43 to 46, wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR2 includes the amino acid sequence set forth in SEQ ID NO: 104, FR3 includes the amino acid sequence set forth in SEQ ID NO: 124, FR4 Comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 120 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

48. The polypeptide of embodiment 47, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 83, 104, 124, 131, 79, 100, A polypeptide having an amino acid sequence having an identity to the polypeptide.

49. The polypeptide of any of embodiments 41-48, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 58 or a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 58.

50. The method of any one of embodiments 42 to 46, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity to SEQ ID NO: 218 or SEQ ID NO: 218, wherein said first immunoglobulin Wherein the single variable domain comprises an amino acid sequence as set forth in SEQ ID NO: 216 or an amino acid sequence having at least 80% amino acid identity to SEQ ID NO: 216.

51. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, Wherein the first immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 161, and 181 or at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence, (163E3 / 127D1) comprising at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence of SEQ ID NO: 161 or SEQ ID NO: 181.

52. The method of embodiment 51 comprising the structure set forth in embodiment 14 wherein CDR1 in the second immunoglobulin of said single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 146 and CDR2 has the amino acid sequence set forth in SEQ ID NO: CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 186, CDR4 in the first immunoglobulin of said single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 141, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 161, and CDR6 Lt; RTI ID = 0.0 &gt; 181. &lt; / RTI &gt;

53. The method of embodiment 52 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 176, 166, 186, 141, 161 or 181 &Lt; / RTI &gt;

54. The polypeptide of embodiment 52 or 53, wherein the amino acid sequence is different only in conservative amino acid changes from those shown in SEQ ID NO: 146, 166, 186, 141, 161 or 181.

55. The method according to any one of embodiments 52-54, wherein the framework regions in each monomer of the single variable domain comprise one or more amino acids at amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 Wherein the polypeptide comprises a Mark residue.

56. The polypeptide according to any one of embodiments 52 to 55, wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 125, FR4 Comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 79, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 100, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 120 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

57. The polynucleotide of embodiment 56 wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 84, 105, 125, 131, 79, 100, Wherein the polypeptide has an amino acid sequence that is identical to the polypeptide.

58. The polypeptide of any of embodiments 51-57, comprising a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 59 or the amino acid sequence of SEQ ID NO: 59.

59. The method of any one of embodiments 51-58, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217, wherein said first immunoglobulin Wherein the single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 216 or SEQ ID NO: 216.

60. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOs: 146, 166 and 186 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, At least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the first immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171, and 191, , At least one CDR comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence of 171 or 191 (163E3 / 54B12).

61. The method of embodiment 60 comprising the structure set forth in embodiment 14 wherein CDR1 in said second immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 146 and CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 166 CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 186, CDR4 in said first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, 191. &lt; / RTI &gt;

62. The method of embodiment 61 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 146, 166, 186, 151, 171 or 191 &Lt; / RTI &gt;

63. The polypeptide of embodiment 61 or 62, wherein the amino acid sequence differs from that shown in SEQ ID NO: 146, 166, 186, 151, 171 or 191 only in conservative amino acid changes.

64. The method of any one of embodiments 61-63, wherein the framework regions within each monomer of the single variable domain are selected from amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

Wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 125, and FR4 Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

66. The method of embodiment 65 wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 are mixed with an amino acid sequence as set forth in any of SEQ ID NOs: 84, 105, 125, 131, 89, 110, A polypeptide having an amino acid sequence having an identity to the polypeptide.

67. The polypeptide according to any one of embodiments 60-66, comprising a polypeptide having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62.

68. The method of any one of embodiments 60-64, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217, wherein said first immunoglobulin Wherein the single variable domain comprises the amino acid sequence as set forth in SEQ ID NO: 219 or an amino acid sequence having at least 80% amino acid identity with SEQ ID NO: 219.

69. The method according to any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, At least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the first immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 151, 171, and 191, , At least one CDR comprising an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 171 or 191, (163D2 / 54B12).

70. The method of embodiment 69 comprising the structure set forth in embodiment 14, wherein said second immunoglobulin single variable domain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 145, said CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 165 , CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 185, CDR4 in said first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, &Lt; / RTI &gt;

Wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 is at least 80% amino acid identical to any one of the amino acid sequences set forth in SEQ ID NO: 145, 165, 185, 151, 171, &Lt; / RTI &gt;

72. The polypeptide of embodiment 70 or 71, wherein the amino acid sequence differs from that shown in SEQ ID NO: 145, 165, 185, 151, 171 or 191 only in conservative amino acid changes.

73. The method according to any one of embodiments 70 to 72, wherein the framework regions within each monomer of the single variable domain are selected from amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

74. The polypeptide according to any one of embodiments 70 to 73, wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR2 includes the amino acid sequence set forth in SEQ ID NO: 104, FR3 includes the amino acid sequence set forth in SEQ ID NO: 124, FR4 Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

75. The polypeptide of embodiment 74, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOs: 83, 104, 124, 131, 89, Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;

76. The polypeptide of any of embodiments 69 to 75, comprising a polypeptide having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63.

77. The method of any one of embodiments 69-73, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 218 or SEQ ID NO: 218, wherein said first immunoglobulin Wherein the single variable domain comprises the amino acid sequence as set forth in SEQ ID NO: 219 or an amino acid sequence having at least 80% amino acid identity with SEQ ID NO: 219.

78. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said first immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 And at least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the second immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 166, and 186, (2B2 / 163E3) comprising at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence of SEQ ID NOs: 166 and 186.

79. The method of embodiment 78 comprising the structure set forth in embodiment 14 wherein the CDR1 in the first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 147 and the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 187, CDR4 in said second immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 166, 186. &lt; / RTI &gt;

80. The method of embodiment 79 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 147, 167, 187, 146, 166 or 186 &Lt; / RTI &gt;

81. The polypeptide of embodiment 79 or 80, wherein the amino acid sequence differs from that shown in SEQ ID NO: 147, 167, 187, 146, 166 or 186 only in conservative amino acid changes.

82. The method of any one of embodiments 79-81 wherein the framework regions within each monomer of the single variable domain are selected from the group consisting of amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

Wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 126, and FR4 Comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 84, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 105, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 125 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

84. The polypeptide of embodiment 83, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOS: 85, 106, 126, 131, 84, Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;

85. The polypeptide according to any one of embodiments 78 to 84, comprising a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of SEQ ID NO: 64.

86. The method of any one of embodiments 78-82, wherein said first immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 213 or 214, or SEQ ID NO: 213 or 214, Wherein the second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 217 or SEQ ID NO: 217.

87. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said first immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOs: 147, 167 and 187 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187 Wherein the second immunoglobulin variable domain comprises at least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence and wherein the second immunoglobulin variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145, 165 and 185, And at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with an amino acid sequence of 185 (2B2 / 163D2).

88. The method of embodiment 87 comprising the structure set forth in embodiment 14 wherein CDR1 in the first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 147 and CDR2 comprises the amino acid sequence set forth in SEQ ID NO: CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 187, CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 145 in said second immunoglobulin single variable domain, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, 185 &lt; / RTI &gt;

89. The method of embodiment 88 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 147, 167, 187, &Lt; / RTI &gt;

90. The polypeptide of embodiment 88 or 89, wherein the amino acid sequence differs from that shown in SEQ ID NO: 147, 167, 187, 145, 165 or 185 only in conservative amino acid changes.

91. In any one of embodiments 88-90, the framework regions within each monomer of a single variable domain are selected from the group consisting of amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

Wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 126, and FR4 Comprises the amino acid sequence set forth in SEQ ID NO: 131, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 83, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 104, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 124, and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

93. The polypeptide of embodiment 94, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% amino acid identity with the amino acid sequence set forth in any of SEQ ID NOS: 85, 106, 126, 131, 83, 104 or 124 Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;

94. The polypeptide according to any one of embodiments 87-93, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 65 or a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 65.

95. The method of any one of embodiments 87-91, wherein said first immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the amino acid sequence set forth in SEQ ID NO: 213 or 214, or SEQ ID NO: 213 or 214, Wherein the second immunoglobulin single variable domain comprises the amino acid sequence as set forth in SEQ ID NO: 218 or an amino acid sequence having at least 80% identity with SEQ ID NO: 218.

96. The method of any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 Wherein the first immunoglobulin single variable domain comprises at least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the first immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147, 167 and 187, 167 and 187 comprising at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 167 and 187. (97A9 / 2B2).

97. The method of embodiment 96 comprising the structure set forth in embodiment 14 wherein CDR1 in said second immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 143 and CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163 CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 183 and CDR4 in said first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 147, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 167, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: &Lt; / RTI &gt;

98. The method of embodiment 97 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 143, 163, 183, 147, 167 or 187 &Lt; / RTI &gt;

99. The polypeptide of embodiment 97 or 98, wherein the amino acid sequence differs only in conservative amino acid changes from those shown in SEQ ID NO: 143, 163, 183, 147, 167 or 187.

100. In any one of embodiments 97-99, the framework region within each monomer of a single variable domain is at amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

101. The method according to any one of embodiments 97-100, wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 122, FR4 Wherein FR5 comprises the amino acid sequence set forth in SEQ ID NO: 133, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 85, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 106, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 126, and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

102. The polypeptide of embodiment 101, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 have at least 80% sequence identity with the amino acid sequence set forth in any of SEQ ID NOs: 81, 102, 122, 133, 85, 106, A polypeptide modified to have an amino acid sequence having amino acid identity.

103. The polypeptide according to any one of embodiments 96-102, comprising a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47.

104. The method of any one of embodiments 96-100, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the sequence or amino acid sequence set forth in SEQ ID NO: 215, Wherein the globulin single variable domain comprises an amino acid sequence having at least 80% identity to the sequence of amino acids set forth in SEQ ID NOS: 213 and 214, or SEQ ID NO: 213 or 214, respectively.

105. The method according to any one of embodiments 3-14 or embodiments 22-39, wherein said second immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOs: 143, 163 and 183 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 143, 163 and 183 Wherein the first immunoglobulin single variable domain comprises at least one CDR comprising an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence, wherein the first immunoglobulin single variable domain is selected from the group consisting of SEQ ID NOS 151, 171, and 191, 171 and 191. The polypeptide (97A9 / 54B12) comprises at least one CDR comprising an amino acid sequence having at least 80% amino acid identity with the amino acid sequence of SEQ ID NOs: 171 and 191.

106. The method of embodiment 105 comprising the structure set forth in embodiment 14 wherein CDR1 in said second immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 143 and CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 163 CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 183, CDR4 in said first immunoglobulin single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 171, 191. &lt; / RTI &gt;

107. The method of embodiment 106 wherein the amino acid sequence of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 has at least 80% amino acid identity with any one of the amino acid sequences set forth in SEQ ID NO: 143, 163, 183, 151, 171 or 191 &Lt; / RTI &gt;

108. The polypeptide of embodiment 106 or 107, wherein the amino acid sequence differs only in conservative amino acid changes from those shown in SEQ ID NO: 143, 163, 183, 151, 171 or 191.

109. The method according to any one of embodiments 106-108, wherein the framework regions in each monomer of the single variable domain are selected from amino acid positions 11, 37, 44, 45, 47, 83, 84, 103, 104 108 comprises at least one hallmark residue.

Wherein FR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, FR2 comprises the amino acid sequence set forth in SEQ ID NO: 102, FR3 comprises the amino acid sequence set forth in SEQ ID NO: 122, FR4 Wherein FR5 comprises the amino acid sequence set forth in amino acid sequence 133, FR5 comprises the amino acid sequence set forth in SEQ ID NO: 89, FR6 comprises the amino acid sequence set forth in SEQ ID NO: 110, FR7 comprises the amino acid sequence set forth in SEQ ID NO: 130 and / Wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.

111. The polynucleotide of embodiment 110, wherein FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 are at least 80% identical to the amino acid sequence shown in any of SEQ ID NOs: 81, 102, 122, 133, 89, 110, A polypeptide modified to have an amino acid sequence having amino acid identity.

112. The polypeptide of any one of embodiments 105-111, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 61 or a polypeptide having at least 80% amino acid identity with the amino acid sequence of SEQ ID NO:

113. The method of any one of embodiments 105-109, wherein said second immunoglobulin single variable domain comprises an amino acid sequence having at least 80% amino acid identity with the sequence or sequence 215 of the amino acid sequence set forth in SEQ ID NO: 215, Wherein the globulin single variable domain comprises the amino acid sequence as set forth in SEQ ID NO: 219 or an amino acid sequence having at least 80% amino acid identity with SEQ ID NO: 219.

114. The polypeptide of any of embodiments 1 to 38 or 42 to 113, comprising a sequence-optimized framework region comprising a partially or fully humanized framework region.

115. A polypeptide that cross-reacts with non-human primate CXCR2 in any one of embodiments 1 to 38 or 42 to 114.

116. The polypeptide of Embodiment 115 wherein the primate is a cynomolgus monkey.

117. The polypeptide of any one of embodiments 1 to 38 or 42 to 116, which does not cross-react with CXCR2 from a non-primate species.

118. The polypeptide of any one of embodiments 1 to 38 or 42 to 117, which does not cross-react with other receptors of the CXC chemokine family.

119. The method according to any one of embodiments 1 to 38 and 114 to 118, wherein said second antigen binding domain comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 8, 9, 10, 11 and 12, Lt; RTI ID = 0.0 &gt; CXCR2 &lt; / RTI &gt; epitope.

120. The polypeptide of embodiment 119, wherein said first antigen binding domain recognizes an epitope comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7.

121. The polypeptide of embodiment 119 or 120, wherein said first antigen binding domain recognizes an epitope comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4.

122. The polypeptide according to any one of embodiments 119 to 121, wherein said second antigen binding domain recognizes an epitope comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5 or 6.

123. In any one of Embodiments 1 to 38 or Embodiments 42 to 122, 10 ^-5 to 10 ^-12 mol / liter or less, preferably 10 ^-7 to 10 ^-12 mol / liter or less, more preferably, A polypeptide capable of specifically binding to human CXCR2 with a dissociation constant (K _D ) of 10 ^-8 to 10 ^-12 moles / liter.

124. In any one of embodiments 1 to 38 or embodiments 42 to 123, a concentration of from 10 ² M ^-1 s ^-1 to about 10 ⁷ M ^-1 s ^-1 , preferably from 10 ³ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , more preferably 10 ⁴ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 , such as 10 ⁵ M ^-1 s ^-1 to 10 ⁷ M ^-1 s ^-1 A polypeptide capable of specifically binding to human CXCR2 at an association rate (k _on rate).

125. A compound according to any one of embodiments 1 to 38 or embodiments 42 to 124, in any one of 1 s ^-1 to 10 ^-6 s ^-1 , preferably 10 ^-2 s ^-1 to 10 ^-6 s ^-1 , To a polypeptide capable of specifically binding to human CXCR2 at a dissociation rate (k _off rate) of 10 ^-3 s ^-1 to 10 ^-6 s ^-1 , such as 10 ^-4 s ^-1 to 10 ^-6 s ^-1 .

126. In any one of embodiments 1 to 38 or embodiments 42 to 125, an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, is specific for human CXCR2 &Lt; / RTI &gt;

127. A polypeptide according to any one of embodiments 1-38 or embodiments 42-126 capable of inhibiting the binding of Gro-a to human CXCR2 with an IC50 of less than 20 nM.

128. A polypeptide according to any one of embodiments 1 to 38 or 42 to 127 capable of inhibiting Gro-α-induced calcium release from RBL cells expressing human CXCR2 with an IC50 of less than 100 nM.

129. The polypeptide according to any one of embodiments 1 to 38 or 42 to 128, capable of inhibiting Gro-a induced accumulation of [ ³⁵ S] GTPγS in human CHO-CXCR2 membranes with an IC50 of less than 50 nM.

130. The polypeptide of any of embodiments 1 to 38 or 42 to 129, wherein the polypeptide is substantially isolated form.

131. The polypeptide of any one of embodiments 1 to 38 and 42 to 130, wherein the polypeptide is a multiple paratovenic construct.

132. A polypeptide modified in any one of embodiments 1 to 38 or 42 to 131 to have an increased in vivo half life compared to the corresponding unmodified amino acid sequence.

133. The method of Embodiment 132, wherein said increased half-life is selected from the group consisting of serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding serum proteins Wherein the polypeptide is provided by one or more binding units.

134. The polypeptide of embodiment 133, wherein said one or more other binding units that provide the polypeptide with an increased half-life are selected from the group consisting of human serum albumin or fragments thereof.

135. The method of claim 133, wherein the one or more other binding units that provide the polypeptide with an increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (e.g., human serum albumin) or serum immunoglobulins (e.g., IgG) Lt; / RTI &gt;

136. The method of embodiment 132, wherein said increased half-life is a domain antibody capable of binding to serum albumin (e.g., human serum albumin) or serum immunoglobulin (e.g., IgG), an amino acid sequence suitable for use as a domain antibody, Wherein the polypeptide is provided by at least one other binding unit selected from the group consisting of an amino acid sequence suitable for use as a single domain antibody, "dAb ", an amino acid sequence suitable for use as a dAb, or a nanobody.

137. The polypeptide of embodiment 134, wherein the polypeptide is an immunoglobulin single variable domain capable of binding to serum albumin (e.g., human serum albumin) or serum immunoglobulin (e.g., IgG).

138. The polypeptide according to any one of embodiments 132-137, wherein the half-life of the corresponding unmodified polypeptide is at least 1.5 times, preferably at least 2 times, such as at least 5 times, such as at least 10 times or more than 20 times Polypeptides with large serum half-lives.

139. In any one of any of embodiments 132-138, greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24 hours relative to the corresponding unmodified polypeptide , 48 or 72 hours. &Lt; / RTI &gt;

140. In any one of embodiments 132-139, at least about 12 hours, preferably at least about 24 hours, more preferably at least about 48 hours, even more preferably at least about 72 hours in human; For example at least about 5 days (e.g. about 5 to 10 days), preferably at least about 9 days (e.g. about 9 to 14 days), more preferably at least about 10 days (e.g. about 10 to 15 days) Polypeptide having a serum half-life of greater than 11 days (e.g., from about 11 to 16 days), more preferably at least about 12 days (e.g., greater than about 12 to 18 days), or greater than 14 days (e.g., from about 14 to 19 days).

141. The pegylated polypeptide of any one of embodiments 1 to 38 or 42 to 140.

142. The corroded polypeptide of any one of embodiments 1-38 or 42-140.

143. The hesated polypeptide of any one of embodiments 1 to 38 or 42 to 140.

144. The method of any one of embodiments 36 or 37 or embodiments 42-143 wherein said additional antigen binding domain has less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as 500 lt; RTI ID = 0.0 &gt; nM. &lt; / RTI &gt;

145. A nucleic acid molecule encoding a polypeptide according to any one of embodiments 1 to 38 or 42 to 144.

146. A nucleic acid molecule encoding an immunoglobulin single variable domain comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 25 to 43, 90 and 213 to 219.

147. The nucleic acid molecule of embodiment 146, comprising a nucleotide sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NOS: 192-211.

148. A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 44 to 69.

149. An expression vector comprising a nucleic acid molecule according to any one of embodiments 145-148.

150. A host cell capable of expressing a polypeptide according to any one of embodiments 1 to 38 or 42 to 149 from a nucleic acid sequence according to any one of embodiments 145 to 148.

151. Monovalent, divalent, polyvalent, double-parathetic or multi-parathetic nano-bodies obtainable by culturing the host cell of embodiment 150.

152. A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1 to 38 or 42 to 144, and a pharmaceutically acceptable carrier or diluent.

153. The polypeptide according to any one of embodiments 1 to 38 or 42 to 144, for use as a medicament.

154. The polypeptide according to any one of embodiments 1 to 38 or 42 to 144, for use in the treatment of chronic obstructive pulmonary disease (COPD) and COPD exacerbation.

155. The method of any one of embodiments 1 to 38 or 42 to 144, wherein the disease is selected from the group consisting of cystic fibrosis, asthma, severe asthma, aggravated asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, Polypeptides for use in the treatment of obstructive bronchiolitis syndrome or bronchopulmonary dysplasia.

156. A compound according to any one of embodiments 1 to 38 or 42 to 144, wherein the compound is selected from the group consisting of atherosclerosis, glomerulonephritis, inflammatory bowel disease (Crohn's disease), angiogenesis and macular degeneration, diabetic retinopathy and diabetic neuropathy. PAH, including familial PAH and related PAHs, chronic inflammatory diseases, including psoriasis, age-related macular degenerative diseases, eyebelt disease, uveitis, idiopathic pulmonary arterial hypertension (PAH) A polypeptide for use in the treatment of rheumatoid arthritis, osteoarthritis, non-small cell carcinoma, colon cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer, solid tumors and metastases, melanoma, hepatocellular carcinoma or ischemic reperfusion injury.

157. A method of treating chronic obstructive pulmonary disease (COPD) or COPD exacerbation comprising administering to a subject an effective amount of a polypeptide according to any one of embodiments 1 to 38 or 42 to 144.

158. A method of treating a condition selected from the group consisting of cystic fibrosis, asthma, severe asthma, aggravated asthma, allergic asthma, acute lung injury, acute &lt; RTI ID = Respiratory distress syndrome, idiopathic pulmonary fibrosis, airway remodeling, obstructive bronchiitis syndrome or bronchopulmonary dysplasia.

159. A method of treating a condition selected from the group consisting of atherosclerosis, glomerulonephritis, inflammatory bowel disease (Crohn's disease), angiogenesis, and macular degeneration, diabetic Psoriasis, age-related macular degeneration diseases, eye-Behcet's disease, uveitis, idiopathic pulmonary arterial hypertension (PAH), familial PAH and related PAHs, including retinopathy and diabetic neuropathy Selected from the group consisting of PAH, chronic inflammatory disease, rheumatoid arthritis, osteoarthritis, non-small cell carcinoma, colon cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer, solid tumor and metastasis, melanoma, hepatocellular carcinoma and ischemic reperfusion injury A method of treating a condition.

Use of a polypeptide according to any one of embodiments 1 to 38 or 42 to 144 in the manufacture of a medicament for the treatment of chronic obstructive pulmonary disease (COPD) or COPD exacerbation.

161. The use of any one of embodiments 1 to 38 in the manufacture of a medicament for the treatment of asthma, severe asthma, aggravation of asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, airway remodeling, obstructive bronchiolitis syndrome or bronchopulmonary dysplasia. Or &lt; RTI ID = 0.0 &gt; 42 &lt; / RTI &gt;

162. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease characterized by a neovascularization including atherosclerosis, glomerulonephritis, inflammatory bowel disease (Crohn's disease), angiogenesis and macular degeneration, diabetic retinopathy and diabetic neuropathy, Chronic inflammatory diseases, rheumatoid arthritis, osteoarthritis, non-small cell carcinoma, colon cancer, pancreatic cancer, esophagus cancer, including age-related macular degenerative diseases, eye disease, uveitis, idiopathic pulmonary arterial hypertension (PAH), familial PAH and related PAH Use of a polypeptide according to any one of Embodiments 1 to 38 or 42 to 144 in the manufacture of a medicament for the treatment of ovarian cancer, breast cancer, solid tumor and metastasis, melanoma, hepatocellular carcinoma or ischemic reperfusion injury.

163. The polypeptide according to any one of embodiments 1 to 38, capable of cross-blocking the binding of a polypeptide according to any one of embodiments 49, 58, 67, 76, 85, 94, 103 or 112 to CXCR2.

164. The method of any of embodiments 39-41, wherein said first and second antigen binding domains comprise any of the features presented in said domain in any one of embodiments 1-22, 32-38, or 42-144. Molecules that do.

165. The molecule of embodiment 164 that exhibits any of the features of any of embodiments 106-135.

166. A composition comprising a polypeptide according to any one of embodiments 49, 58, 67, 76, 85, 94, 103 or 112 and capable of cross-blocking the binding of CXCR2 to at least one immunoglobulin A polypeptide comprising a single variable domain.

167. A kit according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid according to Kabat numbering encoded by the plasmid DSM 23727 deposited with said second immunoglobulin single variable domain Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23726 deposited.

168. The method of embodiment 167, wherein an FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23727 deposited on said second immunoglobulin single variable domain and a plasmid deposited on said first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23726.

169. The polypeptide of embodiment 168, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

170. The method according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein said second immunoglobulin single variable domain is a CDR amino acid according to Kabat numbering encoded by the plasmid DSM 23725 deposited Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23726 deposited.

171. The method of embodiment 170, wherein an FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23725 deposited on said second immunoglobulin single variable domain and a plasmid deposited on said first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23726.

172. The polypeptide of embodiment 171, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

173. The method of any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23725 deposited with said second immunoglobulin single variable domain Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering that is encoded by the deposited plasmid DSM 23728.

174. The recombinant vector of embodiment 173, wherein the FR region having an amino acid sequence according to Kabat numbering and encoded by the plasmid DSM 23725 deposited on the second immunoglobulin single variable domain and the plasmid deposited on the first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering encoded by DSM 23728.

175. The polypeptide of embodiment 174, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

176. A method according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid according to Kabat numbering encoded by the plasmid DSM 23727 deposited with said second immunoglobulin single variable domain Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering that is encoded by the deposited plasmid DSM 23728.

177. The method of embodiment 176, wherein the FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23727 deposited on said second immunoglobulin single variable domain and a plasmid deposited on said first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23728.

178. The polypeptide of embodiment 177, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

179. A kit according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid according to Kabat numbering encoded by the plasmid DSM 23723 deposited with said first immunoglobulin single variable domain Wherein the second immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering encoded by a plasmid DSM 23725 deposited with the second immunoglobulin single variable domain.

180. The method of embodiment 179, wherein an FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23723 deposited on said first immunoglobulin single variable domain and a plasmid deposited on said second immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23725.

181. The polypeptide of embodiment 180, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

182. The method according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23723 deposited with said first immunoglobulin single variable domain Wherein the second immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23727 deposited with the second immunoglobulin single variable domain.

183. The method according to embodiment 182, wherein the FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23723 deposited on said first immunoglobulin single variable domain and the plasmid deposited on said second immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23727.

184. The polypeptide of embodiment 183, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

185. The method according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein the CDR amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23724 deposited with said second immunoglobulin single variable domain Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering encoded by a plasmid DSM 23723 deposited with the first immunoglobulin single variable domain.

186. The method of embodiment 185, wherein an FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23724 deposited on said second immunoglobulin single variable domain and a plasmid deposited on said first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23723.

187. The polypeptide of embodiment 186, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

188. The method according to any one of embodiments 3-14 and 22-39, comprising the structure set forth in embodiment 14, wherein said second immunoglobulin single variable domain is a CDR amino acid according to Kabat numbering encoded by the plasmid DSM 23724 deposited. Wherein the first immunoglobulin single variable domain comprises a CDR amino acid sequence according to Kabat numbering that is encoded by the deposited plasmid DSM 23728.

189. The method of embodiment 188 wherein the FR region having an amino acid sequence according to Kabat numbering encoded by the plasmid DSM 23724 deposited on said second immunoglobulin single variable domain and a plasmid deposited on said first immunoglobulin single variable domain A polypeptide further comprising an FR region having an amino acid sequence according to Kabat numbering coded by DSM 23728.

190. The polypeptide of embodiment 189, wherein said second immunoglobulin single variable domain is linked to said first immunoglobulin single variable domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO: 220.

                         SEQUENCE LISTING <110> NOVARTIS AG   <120> CHEMOKINE RECEPTOR BINDING POLYPEPTIDES <130> PAT054087 <160> 248 <170> PatentIn version 3.5 <210> 1 <211> 360 <212> PRT <213> Homo sapiens <400> 1 Met Glu Asp Phe Asn Met Glu Ser Asp Ser Phe Glu Asp Phe Trp Lys 1 5 10 15 Gly Glu Asp Leu Ser Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Phe             20 25 30 Leu Leu Asp Ala Ala Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Lys         35 40 45 Tyr Phe Val Ile Ile Tyr Ala Leu Val Phe Leu Leu Ser Leu Leu     50 55 60 Gly Asn Ser Leu Val Met Leu Val Ile Leu Tyr Ser Arg Val Gly Arg 65 70 75 80 Ser Val Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ala Asp Leu Leu                 85 90 95 Phe Ala Leu Thr Leu Pro Ile Trp Ala Ala Ser Lys Val Asn Gly Trp             100 105 110 Ile Phe Gly Thr Phe Leu Cys Lys Val Val Ser Leu Leu Lys Glu Val         115 120 125 Asn Phe Tyr Ser Gly Ile Leu Leu Leu Ala Cys Ile Ser Val Asp Arg     130 135 140 Tyr Leu Ala Ile Val Ala Thr Arg Thr Leu Thr Gln Lys Arg Tyr 145 150 155 160 Leu Val Lys Phe Ile Cys Leu Ser Ile Trp Gly Leu Ser Leu Leu Leu                 165 170 175 Ala Leu Pro Val Leu Leu Phe Arg Arg Thr Val Tyr Ser Ser Asn Val             180 185 190 Ser Pro Ala Cys Tyr Glu Asp Met Gly Asn Asn Thr Ala Asn Trp Arg         195 200 205 Met Leu Leu Arg Ile Leu Pro Gln Ser Phe Gly Phe Ile Val Pro Leu     210 215 220 Leu Ile Met Leu Phe Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Lys 225 230 235 240 Ala His Met Gly Gln Lys His Arg Ala Met Arg Val Ile Phe Ala Val                 245 250 255 Val Leu Ile Phe Leu Leu Cys Trp Leu Pro Tyr Asn Leu Val Leu Leu             260 265 270 Ala Asp Thr Leu Met Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Arg         275 280 285 Arg Asn His Ile Asp Arg Ala Leu Asp Ala Thr Glu Ile Leu Gly Ile     290 295 300 Leu His Ser Cys Leu Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Lys 305 310 315 320 Phe Arg His Gly Leu Leu Lys Ile Leu Ale Ile His Gly Leu Ile Ser                 325 330 335 Lys Asp Ser Leu Pro Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Ser             340 345 350 Ser Gly His Thr Ser Thr Thr Leu         355 360 <210> 2 <211> 344 <212> PRT <213> Homo sapiens <400> 2 Met Glu Asp Leu Ser Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Phe 1 5 10 15 Leu Leu Asp Ala Ala Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Lys             20 25 30 Tyr Phe Val Ile Ile Tyr Ala Leu Val Phe Leu Leu Ser Leu Leu         35 40 45 Gly Asn Ser Leu Val Met Leu Val Ile Leu Tyr Ser Arg Val Gly Arg     50 55 60 Ser Val Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ala Asp Leu Leu 65 70 75 80 Phe Ala Leu Thr Leu Pro Ile Trp Ala Ala Ser Lys Val Asn Gly Trp                 85 90 95 Ile Phe Gly Thr Phe Leu Cys Lys Val Val Ser Leu Leu Lys Glu Val             100 105 110 Asn Phe Tyr Ser Gly Ile Leu Leu Leu Ala Cys Ile Ser Val Asp Arg         115 120 125 Tyr Leu Ala Ile Val Ala Thr Arg Thr Leu Thr Gln Lys Arg Tyr     130 135 140 Leu Val Lys Phe Ile Cys Leu Ser Ile Trp Gly Leu Ser Leu Leu Leu 145 150 155 160 Ala Leu Pro Val Leu Leu Phe Arg Arg Thr Val Tyr Ser Ser Asn Val                 165 170 175 Ser Pro Ala Cys Tyr Glu Asp Met Gly Asn Asn Thr Ala Asn Trp Arg             180 185 190 Met Leu Leu Arg Ile Leu Pro Gln Ser Phe Gly Phe Ile Val Pro Leu         195 200 205 Leu Ile Met Leu Phe Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Lys     210 215 220 Ala His Met Gly Gln Lys His Arg Ala Met Arg Val Ile Phe Ala Val 225 230 235 240 Val Leu Ile Phe Leu Leu Cys Trp Leu Pro Tyr Asn Leu Val Leu Leu                 245 250 255 Ala Asp Thr Leu Met Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Arg             260 265 270 Arg Asn His Ile Asp Arg Ala Leu Asp Ala Thr Glu Ile Leu Gly Ile         275 280 285 Leu His Ser Cys Leu Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Lys     290 295 300 Phe Arg His Gly Leu Leu Lys Ile Leu Ale Ile His Gly Leu Ile Ser 305 310 315 320 Lys Asp Ser Leu Pro Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Ser                 325 330 335 Ser Gly His Thr Ser Thr Thr Leu             340 <210> 3 <211> 355 <212> PRT <213> Macaca fascicularis <400> 3 Met Gln Ser Phe Asn Phe Glu Asp Phe Trp Glu Asn Glu Asp Phe Ser 1 5 10 15 Asn Tyr Ser Tyr Ser Ser Asp Leu Pro Pro Ser Leu Pro Asp Val Ala             20 25 30 Pro Cys Arg Pro Glu Ser Leu Glu Ile Asn Lys Tyr Phe Val Val Ile         35 40 45 Ile Tyr Ala Leu Val Leo Le Leu Le Leu Gly Asn Ser Leu Val     50 55 60 Met Leu Val Ile Leu His Ser Arg Val Gly Arg Ser Ile Thr Asp Val 65 70 75 80 Tyr Leu Leu Asn Leu Ala Met Ala Asp Leu Leu Phe Ala Leu Thr Leu                 85 90 95 Pro Ile Trp Ala Ala Lys Val Asn Gly Trp Ile Phe Gly Thr Phe             100 105 110 Leu Cys Lys Val Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Ser Gly         115 120 125 Ile Leu Leu Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Val     130 135 140 His Ala Thr Arg Thr Leu Thr Gln Lys Arg Tyr Leu Val Lys Phe Val 145 150 155 160 Cys Leu Ser Ile Trp Ser Leu Ser Leu Leu                 165 170 175 Leu Phe Arg Arg Thr Val Tyr Leu Thr Tyr Ile Ser Pro Val Cys Tyr             180 185 190 Glu Asp Met Gly Asn Asn Thr Ala Lys Trp Arg Met Val Leu Arg Ile         195 200 205 Leu Pro Gln Thr Phe Gly Phe Ile Leu Pro Leu Leu Ile Met Leu Phe     210 215 220 Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Lys Ala His Met Gly Gln 225 230 235 240 Lys His Arg Ala Met Arg Val Ile Phe Ala Val Val Leu Ile Phe Leu                 245 250 255 Leu Cys Trp Leu Pro Tyr His Leu Val Leu Leu Ala Asp Thr Leu Met             260 265 270 Arg Thr Arg Leu Ile Asn Glu Thr Cys Gln Arg Arg Asn Asn Ile Asp         275 280 285 Gln Ala Leu Asp Ala Thr Glu Ile Leu Gly Ile Leu His Ser Cys Leu     290 295 300 Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Lys Phe Arg His Gly Leu 305 310 315 320 Leu Lys Ile Leu Ala Thr His Gly Leu Ile Ser Lys Asp Ser Leu Pro                 325 330 335 Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Ser Ser Gly His Thr Ser             340 345 350 Thr Thr Leu         355 <210> 4 <211> 14 <212> PRT <213> Macaca fascicularis <400> 4 Met Gln Ser Phe Asn Phe Glu Asp Phe Trp Glu Asn Glu Asp 1 5 10 <210> 5 <211> 18 <212> PRT <213> Macaca fascicularis <400> 5 Cys Thr Leu Met Arg Thr Arg Leu Ile Asn Glu Thr Leu Gln Arg Arg 1 5 10 15 Asn Cys          <210> 6 <211> 26 <212> PRT <213> Macaca fascicularis <400> 6 Cys Arg Arg Thr Val Tyr Leu Thr Tyr Ile Ser Pro Val Leu Tyr Glu 1 5 10 15 Asp Met Gly Asn Asn Thr Ala Leu Trp Cys             20 25 <210> 7 <211> 19 <212> PRT <213> Homo sapiens <400> 7 Met Glu Asp Phe Asn Met Glu Ser Asp Ser Phe Glu Asp Phe Trp Lys 1 5 10 15 Gly Glu Asp              <210> 8 <211> 31 <212> PRT <213> Homo sapiens <400> 8 Glu Asp Leu Ser Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Phe Leu 1 5 10 15 Leu Asp Ala Ala Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Lys             20 25 30 <210> 9 <211> 26 <212> PRT <213> Homo sapiens <400> 9 Phe Arg Arg Thr Val Tyr Ser Ser Asn Val Ser Pro Ala Cys Tyr Glu 1 5 10 15 Asp Met Gly Asn Asn Thr Ala Asn Trp Arg             20 25 <210> 10 <211> 26 <212> PRT <213> Homo sapiens <400> 10 Cys Arg Arg Thr Val Tyr Ser Ser Asn Val Ser Pro Ala Leu Tyr Glu 1 5 10 15 Asp Met Gly Asn Asn Thr Ala Asn Trp Cys             20 25 <210> 11 <211> 18 <212> PRT <213> Homo sapiens <400> 11 Asp Thr Leu Met Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Arg Arg 1 5 10 15 Asn His          <210> 12 <211> 18 <212> PRT <213> Homo sapiens <400> 12 Cys Thr Leu Met Arg Thr Gln Val Ile Gln Glu Thr Leu Glu Arg Arg 1 5 10 15 Asn Cys          <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 13 ggctgagctg ggtggtcctg g 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 14 ggctgagttt ggtggtcctg g 21 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 15 ggtacgtgct gttgaactgt tcc 23 <210> 16 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 16 catttgagtt ggcctagccg gccatggcag aggtgcagct ggtggagtct ggggg 55 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 17 tgtaaaacga cggccagt 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 18 caggaaacag ctatgacc 18 <210> 19 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 19 tcagtaacct ggatcccccg ccaccgctgc ctccaccgcc gctacccccg ccaccgctgc 60 ctccaccgcc tgaggagacg gtgacctg 88 <210> 20 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 20 aggttactga ggatccggcg gtggaggcag cggaggtggg ggctctggtg gcgggggtag 60 cgaggtgcag ctggtggagt ctgg 84 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 21 gaggtgcaat tggtggagtc tggg 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 22 tgaggagacg gtgacctggg tccc 24 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 23 tcttggatcc gaggtgcagc tggtggagtc tggg 34 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 24 accgcctccg gaggagaccg tgacctgggt ccc 33 <210> 25 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val         35 40 45 Ser Ala Ile Asn Aly Gly Gly Asp Ser Thr Tyr Tyr Ala Asp Pro Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asn Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Thr Val Arg Gly Thr Ala Arg Asp Leu Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Gln Val Thr Val Ser Ser         115 <210> 26 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 26 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Arg Ile Gly Ser Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Val Ser Gly Gln Gln Arg Glu Leu Val         35 40 45 Ala Val Ser Arg Ser Gly Gly Ser Thr Asp Ile Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr                 85 90 95 Ala His Thr Ser Ser Tyr Ser Asn Trp Arg Val Tyr Asn Asn Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 27 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Ile Gly Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Val Ile Thr Ser Gly Gly Arg Ile Asp Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn                 85 90 95 Val Glu Thr Val Val Gly Ala Val Tyr Trp Gly Gln Gly Thr Gln Val             100 105 110 Thr Val Ser Ser         115 <210> 28 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Gly Asn Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val         35 40 45 Ala Lys Ile Thr Arg Gly Gly Ala Ile Thr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ala Arg Asp Asn Ile Leu Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn                 85 90 95 Val Asp Gly Gly Pro Ser Gln Asn Tyr Trp Gly Gln Gly Thr Gln Val             100 105 110 Thr Val Ser Ser         115 <210> 29 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val         35 40 45 Ser Cys Ile Ser Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Tyr Trp Gly Leu Thr Leu Arg Leu Trp Met Pro Pro His Arg             100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 125 <210> 30 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Leu Ile Phe Arg Leu Ser             20 25 30 Gly Met Ala Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Trp Val         35 40 45 Ala Val Leu Thr Lys Asp Gly Thr Leu His Tyr Ala Asp Pro Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Trp Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Asn                 85 90 95 Thr Gly Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             100 105 110 <210> 31 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Gly Thr Ile Arg             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Leu Ile Thr Ser Thr Gly Arg Ile Asn Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Gly Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn                 85 90 95 Ile Glu Thr Leu Arg Arg Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser         115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Asn Lys Ser Gly Gly Asn Thr His Tyr Ala Gly Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Arg Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Ser Arg Thr Asn Pro Lys Pro Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Gln Val Thr Val Ser Ser         115 <210> 33 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Arg Ser Ser Ser Ser Ser Ser             20 25 30 Ala Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Gly Ile Ser Trp Gly Gly Asp Asn Ser Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Gln Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Arg Tyr Arg Gly Gly Ala Ala Val Ala Gly Trp Glu Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 34 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Ala Tyr Tyr             20 25 30 Thr Val Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Gly Ile         35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Arg Arg Thr Asp Cys Lys Lys Gly Arg Val Gly Ser Gly             100 105 110 Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 35 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 35 Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Asn Tyr Tyr             20 25 30 Val Met Ala Trp Phe Arg Gln Ala Gln Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Ser Thr Arg Gly Ser Met Thr Lys Tyr Ser Asp Ser Val     50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Pro Arg Gly Ser Ser Trp Ser Phe Ser Ser Gly Gly Tyr             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 125 <210> 36 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg Leu Ser             20 25 30 Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp Val         35 40 45 Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser             100 105 110 <210> 37 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser         115 <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Ser Ser Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp His Trp Gly Gln             100 105 110 Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 39 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 125 <210> 40 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Leu             20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ala Phe Val         35 40 45 Ala Ala Leu Thr Arg Asn Gly Gly Tyr Arg Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Val Ala Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Ser Leu Ser Gly Ser Asp Tyr Leu Gly Thr Asn Leu Asp             100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 41 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 42 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 43 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser         115 <210> 44 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> multivalent anti-CXCR2 Nanobodies <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly                 165 170 175 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile             180 185 190 Asn Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu         195 200 205 Val Ala Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val     210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 245 250 255 Asn Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg             260 265 270 Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 285 <210> 45 <211> 257 <212> PRT <213> Artificial Sequence <220> <223> multivalent anti-CXCR2 Nanobodies <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg Leu Ser             20 25 30 Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp Val         35 40 45 Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly             100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg                 165 170 175 Leu Ser Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu             180 185 190 Trp Val Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile     210 215 220 Tyr Leu His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser                 245 250 255 Ser      <210> 46 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly                 165 170 175 Ser Ile Val Arg Ile Asn Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly             180 185 190 Lys Gln Arg Glu Leu Val Ala Asp Ile Thr Ser Gly Gly Asn Ile Asn         195 200 205 Tyr Ile Asp Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr     210 215 220 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Asn Ala Glu Ile Val Val Leu Val Gly Val Trp                 245 250 255 Thr Gln Arg Ala Arg Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val             260 265 270 Thr Val Ser Ser         275 <210> 47 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly                 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile             180 185 190 Asn Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu         195 200 205 Val Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val     210 215 220 Lys Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 245 250 255 Met Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val             260 265 270 Thr Val Ser Ser         275 <210> 48 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Val Arg Leu Ser Cys Val Ala Ser Gly                 165 170 175 Ile Ile Phe Arg Leu Ser Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly             180 185 190 Lys Ala Arg Glu Trp Val Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn         195 200 205 Tyr Ala Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala     210 215 220 Lys Asn Thr Ile Tyr Leu His Met Asp Met Leu Lys Pro Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Ala Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln                 245 250 255 Val Thr Val Ser Ser             260 <210> 49 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg Leu Ser             20 25 30 Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp Val         35 40 45 Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly             100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr                 165 170 175 Ile Asn Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu             180 185 190 Leu Val Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met     210 215 220 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Met Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln                 245 250 255 Val Thr Val Ser Ser             260 <210> 50 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly                 165 170 175 Gly Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg Leu             180 185 190 Ser Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp         195 200 205 Val Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val     210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr 225 230 235 240 Leu His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 245 250 255 Ala Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 51 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg Leu Ser             20 25 30 Ala Leu Gly Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp Val         35 40 45 Ala Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 His Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Ser Gly Lys Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly             100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg                 165 170 175 Ile Asn Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu             180 185 190 Leu Val Ala Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val     210 215 220 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Asn Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala                 245 250 255 Arg Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 52 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu         115 120 125 Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly Ser Leu Arg Leu     130 135 140 Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn Ala Met Gly Trp 145 150 155 160 Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Val Arg Arg Thr                 165 170 175 Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys Gly Arg Phe Thr             180 185 190 Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu Gln Met Asn Ser         195 200 205 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Leu Asp Asp Arg     210 215 220 Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 225 230 235 <210> 53 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Asn Gly Gly Arg Val         195 200 205 Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn     210 215 220 Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr                 245 250 255 Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val             260 265 270 Ser Ser          <210> 54 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly                 165 170 175 Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Arg Ser Gly Gly Ser         195 200 205 Ala Tyr Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp     210 215 220 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser                 245 250 255 Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 55 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser                 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr Ala             180 185 190 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala         195 200 205 Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val Lys     210 215 220 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 225 230 235 240 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 245 250 255 Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala Tyr             260 265 270 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 <210> 56 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser                 165 170 175 Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn Ala             180 185 190 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala         195 200 205 Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Ala     210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 245 250 255 Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp Gly             260 265 270 Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 <210> 57 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln 145 150 155 160 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg                 165 170 175 Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn Ala Met Gly             180 185 190 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile         195 200 205 Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Ala Lys Gly     210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala                 245 250 255 Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp Gly Gln Gly             260 265 270 Thr Gln Val Thr Val Ser Ser         275 <210> 58 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu Ser                 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys Val             180 185 190 Met Gly Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val Ala         195 200 205 Ala Ile Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys Gly     210 215 220 Arg Phe Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Val                 245 250 255 Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val             260 265 270 Ser Ser          <210> 59 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln 145 150 155 160 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu Ser Leu Arg                 165 170 175 Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys Val Met Gly             180 185 190 Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val Ala Ala Ile         195 200 205 Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys Gly Arg Phe     210 215 220 Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Val Asn Ile                 245 250 255 Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 60 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln 145 150 155 160 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg                 165 170 175 Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr Ala Met Gly             180 185 190 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile         195 200 205 Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val Lys Gly Arg     210 215 220 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met 225 230 235 240 Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp                 245 250 255 Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala Tyr Trp Gly             260 265 270 Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 <210> 61 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly         115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly                 165 170 175 Gly Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Arg Ile             180 185 190 Asn Thr Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu         195 200 205 Val Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val     210 215 220 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 245 250 255 His Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly             260 265 270 Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 <210> 62 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 62 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln 145 150 155 160 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Thr                 165 170 175 Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Arg Ile Asn Thr Met Gly             180 185 190 Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Arg         195 200 205 Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys Gly Arg Phe     210 215 220 Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala Gly Thr                 245 250 255 Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln Gly Thr Gln             260 265 270 Val Thr Val Ser Ser         275 <210> 63 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser                 165 170 175 Leu Thr Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Arg Ile Asn Thr             180 185 190 Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala         195 200 205 Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys Gly     210 215 220 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala                 245 250 255 Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln Gly             260 265 270 Thr Gln Val Thr Val Ser Ser         275 <210> 64 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 64 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly                 165 170 175 Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Arg Ser Gly Gly Ser         195 200 205 Ala Tyr Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp     210 215 220 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser                 245 250 255 Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 65 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Val Glu Gly Gly Gly Gly 145 150 155 160 Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Asn Gly Gly Arg Val         195 200 205 Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn     210 215 220 Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr                 245 250 255 Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val             260 265 270 Ser Ser          <210> 66 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln 145 150 155 160 Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly Ser Leu Arg                 165 170 175 Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn Ala Met Gly             180 185 190 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Val Arg Arg         195 200 205 Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys Gly Arg Phe     210 215 220 Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Leu Asp Asp                 245 250 255 Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 270 <210> 67 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly Ser                 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn Ala             180 185 190 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Val         195 200 205 Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys Gly     210 215 220 Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Leu                 245 250 255 Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val             260 265 270 Ser Ser          <210> 68 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln Leu Val 145 150 155 160 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser                 165 170 175 Cys Val Ala Ser Gly Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe             180 185 190 Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile Thr Trp         195 200 205 Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Ala Lys Gly Arg Phe     210 215 220 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly                 245 250 255 Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Gln             260 265 270 Val Thr Val Ser Ser         275 <210> 69 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Sequences of multivalent anti-CXCR2 Nanobodies <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Gln Leu Val 145 150 155 160 Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser                 165 170 175 Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe             180 185 190 Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile Thr Trp         195 200 205 Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr     210 215 220 Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser 225 230 235 240 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp                 245 250 255 Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly             260 265 270 Thr Gln Val Thr Val Ser Ser         275 <210> 70 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> fr1 <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser             20 25 30 <210> 71 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> fr1 <400> 71 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu Ser Gly Arg Ile Gly Ser             20 25 30 <210> 72 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp             20 25 30 <210> 73 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Leu Ile Phe Arg             20 25 30 <210> 74 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser             20 25 30 <210> 75 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Ser Phe Ser             20 25 30 <210> 76 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Ala             20 25 30 <210> 77 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 77 Lys Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Asn             20 25 30 <210> 78 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Val Arg Leu Ser Cys Val Ala Ser Gly Ile Ile Phe Arg             20 25 30 <210> 79 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp             20 25 30 <210> 80 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 80 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Ser Ser Phe Arg             20 25 30 <210> 81 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Val Arg             20 25 30 <210> 82 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser             20 25 30 <210> 83 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser             20 25 30 <210> 84 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ile Phe Ser             20 25 30 <210> 85 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Glu Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr             20 25 30 <210> 86 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Ile Gly Thr             20 25 30 <210> 87 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Gly Asn             20 25 30 <210> 88 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 88 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Gly Thr             20 25 30 <210> 89 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Arg             20 25 30 <210> 90 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> monovalent anti-CXCR2 Nanobodies <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 91 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 91 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val Ser 1 5 10 <210> 92 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 92 Trp Tyr Arg Gln Val Ser Gly Gln Gln Arg Glu Leu Val Ala 1 5 10 <210> 93 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 93 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ser 1 5 10 <210> 94 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 94 Trp Tyr Arg Gln Ala Pro Gly Arg Gln Arg Glu Trp Val Ala 1 5 10 <210> 95 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 95 Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 96 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 96 Trp Leu Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 97 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 97 Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Gly Ile Ser 1 5 10 <210> 98 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 98 Trp Phe Arg Gln Ala Gln Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 99 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 99 Trp Thr Arg Gln Gly Pro Gly Lys Ala Arg Glu Trp Val Ala 1 5 10 <210> 100 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 100 Trp Tyr Arg Gln Pro Pro Gly Lys Gln Arg Glu Gly Val Ala 1 5 10 <210> 101 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 101 Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 102 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 102 Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 103 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 103 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ala Phe Val Ala 1 5 10 <210> 104 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 104 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 105 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 105 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 106 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 106 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Val 1 5 10 <210> 107 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 107 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 108 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 108 Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 109 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 109 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 110 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 110 Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 111 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 111 Arg Phe Thr Ile Ser Arg Asp Asn Asn Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Thr             20 25 30 <210> 112 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 112 Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Ala             20 25 30 <210> 113 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 113 Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 114 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 114 Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Trp Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Asn Thr             20 25 30 <210> 115 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 115 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Arg Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 116 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 116 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Gln Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 117 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 117 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 118 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 118 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu His 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 119 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 119 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu His 1 5 10 15 Met Asp Met Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser             20 25 30 <210> 120 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 120 Arg Phe Thr Ile Ser Lys Ala Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Val             20 25 30 <210> 121 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 121 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala             20 25 30 <210> 122 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 122 Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala             20 25 30 <210> 123 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 123 Arg Phe Thr Ile Ser Arg Asp Val Ala Lys Lys Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 124 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 124 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 125 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 125 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 126 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 126 Arg Phe Thr Ile Ser Ala Asp Ile Ala Lys Lys Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Leu             20 25 30 <210> 127 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 127 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn Val             20 25 30 <210> 128 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 128 Arg Phe Thr Ile Ala Arg Asp Asn Ile Leu Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn Val             20 25 30 <210> 129 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 129 Arg Phe Thr Ile Gly Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Tyr Asn Ile             20 25 30 <210> 130 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 130 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Pro Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala             20 25 30 <210> 131 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 131 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 132 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 132 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 133 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 133 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 134 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 134 Asp Tyr Ala Ile Gly 1 5 <210> 135 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 135 Leu Ser Gly Met Ala 1 5 <210> 136 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 136 Asn Tyr Ala Met Gly 1 5 <210> 137 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 137 Arg Ser Ala Met Gly 1 5 <210> 138 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 138 Tyr Tyr Thr Val Gly 1 5 <210> 139 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 139 Tyr Tyr Val Met Ala 1 5 <210> 140 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 140 Leu Ser Ala Leu Gly 1 5 <210> 141 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 141 Phe Lys Val Met Gly 1 5 <210> 142 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 142 Ile Asn Thr Met Gly 1 5 <210> 143 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 143 Ile Asn Thr Met Gly 1 5 <210> 144 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 144 Ser Leu Ser Met Gly 1 5 <210> 145 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 145 Asp Tyr Ala Met Gly 1 5 <210> 146 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 146 Ser Asn Ala Met Gly 1 5 <210> 147 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 147 Ile Asn Ala Met Gly 1 5 <210> 148 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 148 Ile Asn Ala Met Gly 1 5 <210> 149 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 149 Ile Asn Ala Met Gly 1 5 <210> 150 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 150 Ile Arg Ala Met Gly 1 5 <210> 151 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 151 Ile Asn Thr Met Gly 1 5 <210> 152 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 152 Ala Ile Asn Ala Gly Gly Asp Ser Thr Tyr Ala Asp Pro Val Lys 1 5 10 15 Gly      <210> 153 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 153 Val Ser Arg Ser Gly Gly Ser Thr Asp Ile Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 154 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 154 Cys Ile Ser Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 155 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 155 Val Leu Thr Lys Asp Gly Thr Leu His Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 <210> 156 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 156 Ala Ile Asn Lys Ser Gly Gly Asn Thr His Tyr Ala Gly Ser Val Lys 1 5 10 15 Gly      <210> 157 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 157 Gly Ile Ser Trp Gly Gly Asp Asn Ser Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 158 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 158 Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 159 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 159 Ala Ile Ser Thr Arg Gly Ser Met Thr Lys Tyr Ser Asp Ser Val Gln 1 5 10 15 Gly      <210> 160 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 160 Gly Ile Asn Ser Asp Gly Thr Thr Asn Tyr Ala Asp Pro Val Lys Gly 1 5 10 15 <210> 161 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 161 Ala Ile Arg Leu Ser Gly Asn Met His Tyr Ala Glu Ser Val Lys Gly 1 5 10 15 <210> 162 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 162 Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys Gly 1 5 10 15 <210> 163 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 163 Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ile Asp Ala Val Lys Gly 1 5 10 15 <210> 164 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 164 Ala Leu Thr Arg Asn Gly Gly Tyr Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 165 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 165 Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val Lys 1 5 10 15 Gly      <210> 166 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 166 Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Ala 1 5 10 15 Lys Gly          <210> 167 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 167 Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys Gly 1 5 10 15 <210> 168 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 168 Val Ile Thr Ser Gly Gly Arg Ile Asp Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 169 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 169 Lys Ile Thr Arg Gly Gly Ala Ile Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 170 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 170 Leu Ile Thr Ser Thr Gly Arg Ile Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 171 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 171 Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys Gly 1 5 10 15 <210> 172 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 172 Val Arg Gly Thr Ala Arg Asp Leu Asp Tyr 1 5 10 <210> 173 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 173 His Thr Ser Ser Tyr Ser Asn Trp Arg Val Tyr Asn Asn Asp Tyr 1 5 10 15 <210> 174 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 174 Tyr Trp Gly Leu Thr Leu Arg Leu Trp Met Pro Pro His Arg Tyr Asp 1 5 10 15 Tyr      <210> 175 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 175 Gly Arg Tyr One <210> 176 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 176 Ser Arg Thr Asn Pro Lys Pro Asp Tyr 1 5 <210> 177 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 177 Arg Tyr Arg Gly Gly Ala Ala Val Ala Gly Trp Glu Tyr 1 5 10 <210> 178 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 178 Asp Arg Arg Thr Asp Cys Lys Lys Gly Arg Val Gly Ser Gly Ser 1 5 10 15 <210> 179 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 179 Asp Pro Arg Gly Ser Ser Trp Ser Phe Ser Ser Gly Gly Tyr Asp Tyr 1 5 10 15 <210> 180 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 180 Gly Lys Tyr One <210> 181 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 181 Asn Ile Arg Gly Gln Asp Tyr 1 5 <210> 182 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 182 Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp His 1 5 10 <210> 183 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 183 Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr Gly 1 5 10 15 Asn Tyr          <210> 184 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 184 Asp Ser Leu Ser Gly Ser Asp Tyr Leu Gly Thr Asn Leu Asp Tyr 1 5 10 15 <210> 185 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 185 Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala Tyr 1 5 10 15 <210> 186 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 186 Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr 1 5 10 <210> 187 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 187 Asp Asp Arg Gly Gly Val Tyr 1 5 <210> 188 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 188 Glu Thr Val Val Gly Ala Val Tyr 1 5 <210> 189 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 189 Asp Gly Gly Pro Ser Gln Asn Tyr 1 5 <210> 190 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 190 Glu Thr Leu Arg Arg Asn Tyr 1 5 <210> 191 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR3 <400> 191 Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg 1 5 10 <210> 192 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 192 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt caccttcagt acctactgga tgtattgggt ccgtcaggct 120 ccagggaagg ggctcgactg ggtctcagct attaatgctg gtggtgatag cacatactat 180 gcagaccccg tgaagggccg attcaccatc tccagagaca acaacaagaa cacgctgtat 240 ctgcagatga acagcctgaa acctgaggac acggccctgt attactgtgc gaccgtacga 300 ggcacagctc gtgacttgga ctactggggc caggggaccc aggtcaccgt ctcctca 357 <210> 193 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 193 gaggtgaagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60 tcctgtgcac tctctggaag gatcggcagt atcaacgcca tgggctggta tcgccaggtt 120 tcaggacaac agcgcgagtt ggtcgcagta agcaggagcg gaggtagcac agacattgct 180 gactccgtga agggccgatt caccatctcc agagacaacg gcaagaacac agtgtatctg 240 cagatggaca gcctgaaacc tgaggacacg gccgtctatt actgttatgc tcatacttca 300 cctagata gtctcctca 369 <210> 194 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 194 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactt 60 acctgtgcag cctctggacg catcggcact atcaatgcca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt ggtcgcagtt attactagtg gtggtaggat agactatgca 180 gactccgtga agggccgatt caccatctcc agagacaatg ccaagaacac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccgtctatt actataatgt agaaacggta 300 gtgggtgccg tctactgggg ccaggggacc caggtcaccg tctcctca 348 <210> 195 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 195 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60 tcctgtgcag cctctggaag gatgggcaat atcaatgcca tgggctggta tcgccaggct 120 ccagggaagg agcgcgagtt ggtcgcaaaa attactaggg gtggtgcgat aacctatgca 180 gactccgtga agggccgatt caccatcgcc agagacaata ttctgaacac ggcgtatctg 240 caaatgaacg acctgaaacc tgaggacacg gccgtctatt attataatgt agatgggggg 300 cccagtcaaa actactgggg ccaggggacc caggtcaccg tctcctca 348 <210> 196 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 196 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60 tcctgtgcag cctctggatt cactttcgat gattatgcca taggctggtt ccgccaggcc 120 ccagggaagg agcgtgagag ggtctcatgt attagtggta gtgatggtag cacatactat 180 gcagactccg tcaagggccg attcaccatc tccagtgaca acgccaagaa cacggtgtat 240 ctgcaaatga acaacctgaa acccgaggac acggccgttt attattgtgc agcatattgg 300 ggactaacgc tcaggctatg gatgcccccc caccggtatg actactgggg ccaggggacc 360 caggtcaccg tctcctca 378 <210> 197 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 197 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagcctc 60 tcctgtgcag cctctggact tatcttcaga ctcagtggca tggcctggta tcgccaggct 120 ccggggaggc agcgcgagtg ggtcgcagtg cttaccaaag atggtaccct acactatgca 180 gaccccgtga agggccgatt caccatctcc agaaacaacg ccgagaacac gtggtatctg 240 caaatgaaca gcctgaaacc tgaggacaca gccatctatt actgtaatac gggccgttac 300 tggggccagg ggacccaggt caccgtctcc tca 333 <210> 198 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 198 gggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc actgagactc 60 tcctgtgcag cctctggaac catcggcacg atcagagcca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt ggtcgcattg attactagta ctggtaggat aaactatgca 180 gactccgtga agggccgatt caccattgga agagacaatg ccaagaacac ggcgtatctg 240 caaatgaaca acctgaaacc tgaggacacg gccgtctatt actataatat cgaaacacta 300 cgacgtaact actggggcca ggggacccag gtcaccgtct cctca 345 <210> 199 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 199 gaggtgcagc tggtggagtc tgggggagga ttggtgcagg ctggggggtc tctgagactc 60 tcctgtgcag cctctggacg cacctttagt aactatgcca tgggctggtt ccgccaggcc 120 acagggaagg agcgtgagtt tgtagcagct attaacaaga gtggtgggaa cacacactat 180 gcaggctccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgaa acctagggac acggccgttt attactgtgc agcgtcgcgg 300 actaacccta agcctgacta ctggggccag gggacccagg tcaccgtctc ctca 354 <210> 200 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 200 gaggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggacg ctccttcagt cgcagtgcca tgggctggct ccgccaggct 120 ccagggaagg agcgtgaatt tgtagcaggt attagctggg gtggtgataa ctcatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa caccgtgtct 240 ctacaaatga acagcctgaa acctcaggac acggccgttt attactgtgc agcaagatac 300 cggggaggcg cggcagtagc tggttgggag tactggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 201 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 201 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctccggatc cactttggcc tattataccg taggctggtt ccgccgggcc 120 ccagggaagg agcgcgaggg gatctcatgt attagtagta gtgatggtag cacatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa tacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc ggctgacaga 300 cgtaccgact gtaaaaaggg tagagtcggt tctggttcct ggggccaggg gacccaggtc 360 accgtctcct ca 372 <210> 202 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 202 aaggtgcagc tggtggagtc tgggggaggg ctggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctccggacg cgccttcaat tactatgtca tggcctggtt ccgccaggct 120 caagggaagg agcgtgagtt tgtagcagct attagcacgc gtggtagtat gacaaagtat 180 tcagactccg tgcagggccg gttcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctgcacatga acagcctgaa acctgaggat acggccgttt attactgtgc agcagaccct 300 cgcggcagta gctggtcatt ttcgtccggg ggttatgact actggggcca ggggacccag 360 gtcaccgtct cctca 375 <210> 203 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 203 gggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tgtgagactc 60 tcctgtgtag cctctggaat catcttcaga ctcagtgcgt tgggttggac acgccagggt 120 ccaggaaagg cgcgcgagtg ggtcgcaggt attaacagtg atggtacgac caactacgcc 180 gaccccgtga agggccgatt caccatctcc agagacaacg ccaagaacac gatatatctg 240 cacatggaca tgctgaaacc tgaggatacg gccgtctatt actgtgcctc cggaaagtac 300 cggggccagg ggacccaggt caccgtctcc tca 333 <210> 204 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 204 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggagtc tctgagactc 60 tcctgtgcag cctctggaag caccttcgat ttcaaagtca tgggctggta ccgccagcct 120 ccagggaagc agcgcgaggg ggtcgcagcg attaggctta gtggtaacat gcactatgca 180 gagtccgtga agggccgatt caccatctcc aaagccaacg ccaagaacac agtgtatctg 240 caaatgaaca gcctgagacc tgaggacacg gccgtctatt actgtaaggt gaacattcgg 300 ggccaggact actggggcca ggggacccag gtcaccgtct cctca 345 <210> 205 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 205 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgacgctc 60 tcctgtgcag tctctggaag ctccttcaga atcaatacca tgggctggta ccgccgggct 120 ccagggaagc agcgcgagtt ggtcgcagct cgtgatagag gtggttacat aaactatgta 180 gattccgtga agggccgatt caccgtctcc agagacaacg ccaagcccac aatgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccgtctatt attgtcatgc cgggacccaa 300 gatcggacgg gtcggaattt cgaccactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 206 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 206 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgtag cctctggaag catcgtcaga attaatacca tgggctggta ccgccagact 120 ccagggaagc agcgcgagtt ggtcgcagat attaccagtg gtggtaacat aaactatata 180 gacgccgtga agggccgatt caccatctcc agagacaaca ccaagaacac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccgtctatt actgtaatgc agagatcgtt 300 gttctggtgg gagtttggac ccagcgtgcg cggaccggca actactgggg ccaggggacc 360 caggtcaccg tctcctca 378 <210> 207 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 207 gaggtgcagc tggtggagtc tgggggagga ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctggacg cacgttcagt agcttgtcca tgggctggtt ccgccaggct 120 ccggggaagg agcgtgcctt tgtagcagcg cttactcgaa atggtggtta cagatactat 180 gcagactccg tgaagggccg attcaccatc tccagagacg tcgccaagaa gaccttatat 240 ctgcaaatga acagcctgaa acctgaggac acggccgtct attactgtgc agcagatagt 300 cttagtggta gtgactactt aggaaccaac ctagactact ggggccaggg gacccaggtc 360 accgtctcct ca 372 <210> 208 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 208 gaggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggacg caccttcagt gactatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcagct attacgtgga atggtggtag agtattttat 180 actgcctccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgatgtat 240 ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagataaa 300 gacagacgta ctgactatct agggcacccc gttgcctact ggggccaggg gacccaggtc 360 accgtctcct ca 372 <210> 209 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 209 gaggtgcagc tggtggagtc tgggggagga ttggtgcagc ctgggggctc tctgagactc 60 tcctgtgtag cctctggacg catcttcagt agcaatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcggcc attacctgga ggagtggcgg tagcgcgtac 180 tatgcagact ccgcgaaggg ccgattcacc atctccagag acaacgccaa gaacacggtg 240 tatttgcaaa tgaacagcct gaaacctgag gacacggccg tttattattg tgcagctggt 300 ggtagttcct ggttaagttt tccgccggac tactggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 210 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 210 gaggtgcagc tggtggagtc tgggggagag ttggtgcagc cgggggggtc tctgagactc 60 tcctgtgcag cctctggaag catcttaact atcaatgcca tgggctggta ccgccaggct 120 ccagggaagc agcgcgagtt ggtagtccgt aggactaggg gtggtagtac aacgtatcaa 180 gactccgtga agggccgatt caccatctcc gcagacattg ccaagaaaac gatgtatctc 240 caaatgaaca gcctgaaacc tgaagacacg gccgtctatt actgtatgct agatgaccgt 300 gggggtgtct actggggtca ggggacccag gtcaccgtct cctca 345 <210> 211 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> NUCLEIC ACID ENCODING MONOVALENT NANOBODY <400> 211 gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgacgctc 60 tcctgtgcag tctctggaag caccttcaga atcaatacca tgggctggta ccgccgggct 120 ccagggaagc agcgcgagtt ggtcgcagct cgtgatagag gtggttacat aaactatgta 180 gattccgtga agggccgatt caccgtctcc agagacaacg ccaagcccac aatgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccgtctatt attgtcatgc cgggacccaa 300 gatcggacgg gtcggaattt cgaccgctgg ggccagggga cccaggtcac cgtctcctca 360 <210> 212 <211> 109 <212> PRT <213> Homo sapiens <400> 212 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser             100 105 <210> 213 <211> 115 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 213 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ser Lys Lys Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 214 <211> 115 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 214 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr Ile Asn             20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Val Arg Arg Thr Arg Gly Gly Ser Thr Thr Tyr Gln Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Ile Ser Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu                 85 90 95 Leu Asp Asp Arg Gly Gly Val Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 215 <211> 126 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 215 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Val Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ale Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn                 85 90 95 Ala Glu Ile Val Val Leu Val Gly Val Trp Thr Gln Arg Ala Arg Thr             100 105 110 Gly Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 216 <211> 115 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 216 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 217 <211> 122 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 217 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Ser Asn             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser     50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr                 85 90 95 Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 218 <211> 124 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 218 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr             20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val         35 40 45 Ala Ala Ile Thr Trp Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala             100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 219 <211> 120 <212> PRT <213> Artificial Sequence <220> Amino acid sequences of sequence-optimized variants <400> 219 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 220 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE LINKER <400> 220 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly             20 25 30 Gly Gly Ser         35 <210> 221 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 221 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Leu Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Arg Ser Gly Gly Ser         195 200 205 Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp     210 215 220 Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser                 245 250 255 Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser             260 265 270 <210> 222 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 222 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Leu Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Asn Gly Gly Arg Val         195 200 205 Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn     210 215 220 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr                 245 250 255 Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val             260 265 270 Ser Ser          <210> 223 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 223 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu 145 150 155 160 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser                 165 170 175 Cys Ala Ala Ser Gly Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe             180 185 190 Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile Thr Trp         195 200 205 Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe     210 215 220 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly                 245 250 255 Ser Ser Trp Leu Ser Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Leu             260 265 270 Val Thr Val Ser Ser         275 <210> 224 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 224 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Ser Ser Ser Thr Phe Arg Ile Asn             20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val         35 40 45 Ala Ala Arg Asp Arg Gly Gly Tyr Ile Asn Tyr Val Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Pro Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys His                 85 90 95 Ala Gly Thr Gln Asp Arg Thr Gly Arg Asn Phe Asp Arg Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu 145 150 155 160 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser                 165 170 175 Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe             180 185 190 Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ile Thr Trp         195 200 205 Asn Gly Gly Arg Val Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr     210 215 220 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 225 230 235 240 Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp                 245 250 255 Arg Arg Thr Asp Tyr Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly             260 265 270 Thr Leu Val Thr Val Ser Ser         275 <210> 225 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 225 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Leu Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Ile Phe Ser Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Arg Ser Gly Gly Ser         195 200 205 Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp     210 215 220 Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Gly Ser Ser Trp Leu Ser                 245 250 255 Phe Pro Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser             260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly         275 280 285 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly     290 295 300 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe                 325 330 335 Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu             340 345 350 Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala         355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr     370 375 380 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Thr Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser Gln Gly Thr                 405 410 415 Leu Val Thr Val Ser Ser             420 <210> 226 <211> 426 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 226 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Leu Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Ser Ile Val Arg Ile Asn Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly             180 185 190 Lys Gln Arg Glu Leu Val Ala Asp Ile Thr Ser Gly Gly Asn Ile Asn         195 200 205 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser     210 215 220 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Asn Ala Glu Ile Val Val Leu Val Gly Val Trp                 245 250 255 Thr Gln Arg Ala Arg Thr Gly Asn Tyr Trp Gly Gln Gly Thr Leu Val             260 265 270 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly         275 280 285 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly     290 295 300 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 305 310 315 320 Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser                 325 330 335 Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro             340 345 350 Gly Lys Gly Leu Glu Trp Val Ser Ser Ser Ser Ser Ser Ser Ser Asp         355 360 365 Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp     370 375 380 Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 385 390 395 400 Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser                 405 410 415 Ser Gln Gly Thr Leu Val Thr Val Ser Ser             420 425 <210> 227 <211> 424 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 227 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp Phe Lys             20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val         35 40 45 Ala Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys                 85 90 95 Val Asn Ile Arg Gly Gln Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Glu Gly Glu Leu Leu Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                 165 170 175 Arg Thr Phe Ser Asp Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly             180 185 190 Lys Glu Arg Glu Phe Val Ala Ile Thr Trp Asn Gly Gly Arg Val         195 200 205 Phe Tyr Thr Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn     210 215 220 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Ala Asp Lys Asp Arg Arg Thr Asp Tyr                 245 250 255 Leu Gly His Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val             260 265 270 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly         275 280 285 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     290 295 300 Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 305 310 315 320 Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe                 325 330 335 Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys             340 345 350 Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu         355 360 365 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala     370 375 380 Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 385 390 395 400 Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser Gln                 405 410 415 Gly Thr Leu Val Thr Val Ser Ser             420 <210> 228 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequences of sequence-optimized biparatopic <400> 228 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe             20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ser I Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Thr Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 229 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 229 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Leu Thr             20 25 30 <210> 230 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 230 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Val Arg             20 25 30 <210> 231 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 231 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Asp             20 25 30 <210> 232 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 232 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser             20 25 30 <210> 233 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 233 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser             20 25 30 <210> 234 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 234 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Thr Phe Arg             20 25 30 <210> 235 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 235 Asp Ile Thr Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 236 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 236 Ala Ile Arg Leu Ser Gly Asn Arg His Tyr Ala Glu Ser Val Lys Gly 1 5 10 15 <210> 237 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 237 Ala Ile Thr Trp Arg Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly          <210> 238 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 238 Arg Phe Thr Ile Ser Ala Asp Ile Ser Lys Lys Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu Leu             20 25 30 <210> 239 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 239 Arg Phe Thr Ile Ser Ala Asp Ile Ser Lys Asn Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Leu Leu             20 25 30 <210> 240 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 240 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala             20 25 30 <210> 241 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 241 Arg Phe Thr Ile Ser Arg Ala Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Val             20 25 30 <210> 242 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 242 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 243 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 243 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala             20 25 30 <210> 244 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 244 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Pro Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Ala             20 25 30 <210> 245 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> FR4 <400> 245 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 246 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 246 Thr Tyr Trp Met Tyr 1 5 <210> 247 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 247 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala 1 5 10 <210> 248 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 248 Ile Asn Ala Met Gly 1 5

Claims (121)

A second antigen binding domain that comprises at least two immunoglobulin antigen binding domains and acts on or binds to the chemokine receptor CXCR2 and recognizes a first antigen binding domain that recognizes a first epitope on CXCR2 and a second antigen binding domain that recognizes a second epitope on CXCR2 / RTI &gt;
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR5-CDR4-FR6-CDR5-FR7-
Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &
Wherein FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 comprises a second antigen binding domain and FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 comprises a first antigen binding domain, Wherein FR5-CDR4-FR6-CDR5-FR7-CDR6-FR8 comprises a first antigen binding domain and HLE has an increased in vivo half-life , &Lt; / RTI &gt;
a) the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 237, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 186; or
b) the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 185; or
c) the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 141, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 236, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 181, the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 235, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 183; or
d) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 191, CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 146, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 237, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 186; or
e) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 151, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 191, CDR4 comprises the amino acid sequence set forth in SEQ ID NO: 145, CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 165, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 185; or
f) the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 151, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 171, the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 191, the CDR4 comprises the amino acid sequence set forth in SEQ ID NO: CDR5 comprises the amino acid sequence set forth in SEQ ID NO: 235, and CDR6 comprises the amino acid sequence set forth in SEQ ID NO: 183
Isolated polypeptide. 2. The polypeptide of claim 1, wherein the first antigen binding domain is SEQ ID NO: 216 and the second antigen binding domain is SEQ ID NO: 217. The polypeptide according to claim 1, which comprises SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 226 or SEQ ID NO: 227. 7. The polypeptide according to claim 1, partially or fully humanized. 2. The polypeptide of claim 1, wherein said first antigen binding domain binds to an epitope comprising amino acids F11, F14 and W15 of SEQ ID NO: 1. 6. The polypeptide of claim 5, wherein said epitope is linear. The method of claim 1, wherein said second antigen binding domain binds to an epitope in the outer loop of human CXCR2, and said outer loop comprises amino acid residues 106-120, 184-208 and 274-294 of SEQ ID NO: Polypeptide. 8. The polypeptide of claim 7, wherein the epitope is in amino acid residues 106-120 of SEQ ID NO: 1. 8. The polypeptide according to claim 7, wherein said epitope is in a stereogenic form. 10. The polypeptide of claim 9, wherein said second antigen binding domain binds to an epitope of CXCR2 comprising amino acid residues W112, G115, I282 and T285 of SEQ ID NO: 1. 2. The polypeptide of claim 1, wherein the HLE comprises SEQ ID NO: 228. 2. The polypeptide of claim 1, wherein the polypeptide is SEQ ID NO: 225. 13. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 12. 14. An expression vector comprising a nucleic acid molecule according to claim 13. 14. A host cell comprising an expression vector comprising the nucleic acid molecule according to claim 13 or the nucleic acid molecule according to claim 13. 12. A pharmaceutical composition for treating chronic obstructive pulmonary disease (COPD) or COPD exacerbation comprising an effective amount of a polypeptide according to any one of claims 1 to 12. An effective amount of a polypeptide according to any one of claims 1 to 12 for the treatment of cystic fibrosis, asthma, severe asthma, aggravation of asthma, allergic asthma, acute lung injury, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, A bronchopulmonary syndrome, bronchopulmonary syndrome, or bronchopulmonary dysplasia. 12. A method of treating atherosclerosis, glomerulonephritis, inflammatory bowel disease (Crohn's disease), angiogenesis, and macular degeneration, diabetic retinopathy and diabetic neuropathy comprising an effective amount of a polypeptide according to any one of claims 1 to 12. PAH, including familial PAH and related PAHs, chronic inflammatory diseases, including psoriasis, age-related macular degeneration diseases, eyebelt disease, uveitis, idiopathic pulmonary arterial hypertension (PAH) Inflammatory diseases, rheumatoid arthritis, osteoarthritis, non-small cell carcinoma, colon cancer, pancreatic cancer, esophageal cancer, ovarian cancer, breast cancer, solid tumor and metastasis, melanoma, hepatocellular carcinoma, ischemic reperfusion injury, hemolytic transfusion induced vascular occlusion, Ischemia / reperfusion injury, acute stroke / myocardial infarction, obstructive head injury, post-traumatic inflammation and insulin resistant diabetes mellitus It is a pharmaceutical composition for treating a condition. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete